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Sample records for dsb repair efficiency

  1. An approach to estimate radioadaptation from DSB repair efficiency.

    Science.gov (United States)

    Yatagai, Fumio; Sugasawa, Kaoru; Enomoto, Shuichi; Honma, Masamitsu

    2009-09-01

    In this review, we would like to introduce a unique approach for the estimation of radioadaptation. Recently, we proposed a new methodology for evaluating the repair efficiency of DNA double-strand breaks (DSB) using a model system. The model system can trace the fate of a single DSB, which is introduced within intron 4 of the TK gene on chromosome 17 in human lymphoblastoid TK6 cells by the expression of restriction enzyme I-SceI. This methodology was first applied to examine whether repair of the DSB (at the I-SceI site) can be influenced by low-dose, low-dose rate gamma-ray irradiation. We found that such low-dose IR exposure could enhance the activity of DSB repair through homologous recombination (HR). HR activity was also enhanced due to the pre-IR irradiation under the established conditions for radioadaptation (50 mGy X-ray-6 h-I-SceI treatment). Therefore, radioadaptation might account for the reduced frequency of homozygous loss of heterozygosity (LOH) events observed in our previous experiment (50 mGy X-ray-6 h-2 Gy X-ray). We suggest that the present evaluation of DSB repair using this I-SceI system, may contribute to our overall understanding of radioadaptation.

  2. The impact of heterochromatin on DSB repair.

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    Goodarzi, Aaron A; Noon, Angela T; Jeggo, Penny A

    2009-06-01

    DNA NHEJ (non-homologous end-joining) is the major DNA DSB (double-strand break) repair pathway in mammalian cells. Although NHEJ-defective cell lines show marked DSB-repair defects, cells defective in ATM (ataxia telangiectasia mutated) repair most DSBs normally. Thus NHEJ functions independently of ATM signalling. However, approximately 15% of radiation-induced DSBs are repaired with slow kinetics and require ATM and the nuclease Artemis. DSBs persisting in the presence of an ATM inhibitor, ATMi, localize to heterochromatin, suggesting that ATM is required for repairing DSBs arising within or close to heterochromatin. Consistent with this, we show that siRNA (small interfering RNA) of key heterochromatic proteins, including KAP-1 [KRAB (Krüppel-associated box) domain-associated protein 1], HP1 (heterochromatin protein 1) and HDAC (histone deacetylase) 1/2, relieves the requirement for ATM for DSB repair. Furthermore, ATMi addition to cell lines with genetic alterations that have an impact on heterochromatin, including Suv39H1/2 (suppressor of variegation 3-9 homologue 1/2)-knockout, ICFa (immunodeficiency, centromeric region instability, facial anomalies syndrome type a) and Hutchinson-Guilford progeria cell lines, fails to have an impact on DSB repair. KAP-1 is a highly dose-dependent, transient and ATM-specific substrate, and mutation of the ATM phosphorylation site on KAP-1 influences DSB repair. Collectively, the findings show that ATM functions to overcome the barrier to DSB repair posed by heterochromatin. However, even in the presence of ATM, gamma-H2AX (phosphorylated histone H2AX) foci form on the periphery rather than within heterochromatic centres. Finally, we show that KAP-1's association with heterochromatin is diminished as cells progress through mitosis. We propose that KAP-1 is a critical heterochromatic factor that undergoes specific modifications to promote DSB repair and mitotic progression in a manner that allows localized and transient

  3. Double strand break (DSB) repair in heterochromatin and heterochromatin proteins in DSB repair.

    Science.gov (United States)

    Lemaître, Charlène; Soutoglou, Evi

    2014-07-01

    Chromosomal translocations are a hallmark of cancer cells and they represent a major cause of tumorigenesis. To avoid chromosomal translocations, faithful repair of DNA double strand breaks (DSBs) has to be ensured in the context of high ordered chromatin structure. However, chromatin compaction is proposed to represent a barrier for DSB repair. Here we review the different mechanisms cells use to alleviate the heterochromatic barrier for DNA repair. At the same time, we discuss the activating role of heterochromatin-associated proteins in this process, therefore proposing that chromatin structure, more than being a simple barrier, is a key modulator of DNA repair.

  4. DNA DSB repair pathway choice: an orchestrated handover mechanism.

    Science.gov (United States)

    Kakarougkas, A; Jeggo, P A

    2014-03-01

    DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch from NHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity.

  5. Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

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    Andrew H Lloyd

    Full Text Available DNA double strand breaks (DSBs occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ. We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp deletions occurred at a minority (25-45% of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.

  6. Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

    Science.gov (United States)

    Lloyd, Andrew H; Wang, Dong; Timmis, Jeremy N

    2012-01-01

    DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.

  7. Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest.

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish K; Jeevarajan, Antony S; Pierson, Duane L; Wu, Honglu

    2008-11-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that

  8. The type and yield of ionising radiation induced chromosomal aberrations depend on the efficiency of different DSB repair pathways in mammalian cells

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    Natarajan, Adayapalam T.; Berni, Andrea; Marimuthu, Kodumudi M. [Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo (Italy); Palitti, Fabrizio [Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo (Italy)], E-mail: palitti@unitus.it

    2008-07-03

    In order to evaluate the relative role of two major DNA double strand break repair pathways, i.e., non-homologous end joining (NHEJ) and homologous recombination repair (HRR), CHO mutants deficient in these two pathways and the parental cells (AA8) were X-irradiated with various doses. The cells were harvested at different times after irradiation, representing G{sub 2}, S and G{sub 1} phase at the time of irradiation, The mutant cell lines used were V33 (NHEJ deficient), Irs1SF, 51-D1 (HRR deficient). In addition to parental cell line (AA8), a revertant of V33, namely V33-155 was employed. Both types of mutant cells responded with increased frequencies of chromosomal aberrations at all recovery times in comparison to the parental and revertant cells. Mutant cells deficient in NHEJ were more sensitive in all cell stages in comparison to HRR deficient mutant cells, indicating NHEJ is the major repair pathway for DSB repair through out the cell cycle. Both chromosome and chromatid types of exchange aberrations were observed following G{sub 1} irradiation (16 and 24 h recovery). Interestingly, configurations involving both chromosome (dicentrics) and chromatid exchanges were encountered in G{sub 1} irradiated V33 cells. This may indicate that unrepaired DSBs accumulate in G{sub 1} in these mutant cells and carried over to S phase, where they are repaired by HRR or other pathways such as B-NHEJ (back up NHEJ), which appear to be highly error prone. Both NHEJ and HRR, which share some of the same proteins in their pathways, are involved in the repair of DSBs leading to chromosomal aberrations, but with a major role of NHEJ in all stages of cell cycle.

  9. Cancer TARGETases: DSB repair as a pharmacological target.

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    Samadder, Pounami; Aithal, Rakesh; Belan, Ondrej; Krejci, Lumir

    2016-05-01

    Cancer is a disease attributed to the accumulation of DNA damages due to incapacitation of DNA repair pathways resulting in genomic instability and a mutator phenotype. Among the DNA lesions, double stranded breaks (DSBs) are the most toxic forms of DNA damage which may arise as a result of extrinsic DNA damaging agents or intrinsic replication stress in fast proliferating cancer cells. Accurate repair of DSBs is therefore paramount to the cell survival, and several classes of proteins such as kinases, nucleases, helicases or core recombinational proteins have pre-defined jobs in precise execution of DSB repair pathways. On one hand, the proper functioning of these proteins ensures maintenance of genomic stability in normal cells, and on the other hand results in resistance to various drugs employed in cancer therapy and therefore presents a suitable opportunity for therapeutic targeting. Higher relapse and resistance in cancer patients due to non-specific, cytotoxic therapies is an alarming situation and it is becoming more evident to employ personalized treatment based on the genetic landscape of the cancer cells. For the success of personalized treatment, it is of immense importance to identify more suitable targetable proteins in DSB repair pathways and also to explore new synthetic lethal interactions with these pathways. Here we review the various alternative approaches to target the various protein classes termed as cancer TARGETases in DSB repair pathway to obtain more beneficial and selective therapy.

  10. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair.

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    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-01

    Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

  11. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

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    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  12. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

  13. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

  14. DSB (Im)mobility and DNA repair compartmentalization in mammalian cells.

    Science.gov (United States)

    Lemaître, Charlène; Soutoglou, Evi

    2015-02-13

    Chromosomal translocations are considered as causal in approximately 20% of cancers. Therefore, understanding their mechanisms of formation is crucial in the prevention of carcinogenesis. The first step of translocation formation is the concomitant occurrence of double-strand DNA breaks (DSBs) in two different chromosomes. DSBs can be repaired by different repair mechanisms, including error-free homologous recombination (HR), potentially error-prone non-homologous end joining (NHEJ) and the highly mutagenic alternative end joining (alt-EJ) pathways. Regulation of DNA repair pathway choice is crucial to avoid genomic instability. In yeast, DSBs are mobile and can scan the entire nucleus to be repaired in specialized DNA repair centers or if they are persistent, in order to associate with the nuclear pores or the nuclear envelope where they can be repaired by specialized repair pathways. DSB mobility is limited in mammals; therefore, raising the question of whether the position at which a DSB occurs influences its repair. Here, we review the recent literature addressing this question. We first present the reports describing the extent of DSB mobility in mammalian cells. In a second part, we discuss the consequences of non-random gene positioning on chromosomal translocations formation. In the third part, we discuss the mobility of heterochromatic DSBs in light of our recent data on DSB repair at the nuclear lamina, and finally, we show that DSB repair compartmentalization at the nuclear periphery is conserved from yeast to mammals, further pointing to a role for gene positioning in the outcome of DSB repair. When regarded as a whole, the different studies reviewed here demonstrate the importance of nuclear architecture on DSB repair and reveal gene positioning as an important parameter in the study of tumorigenesis.

  15. Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation

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    O'Neill, Peter; Harper, Jane; Anderson, Jennifer a.; Cucinnota, Francis A.

    2007-01-01

    -radiation but at 0.25 Gy gamma-irradiation the rate of DSB repair is similar in the presence or absence vanillin, thus suggesting the repair of a sub-set of DSBs induced by low dose, low-LET radiation does not require DNA-PK(sub cs). This sub-set of DSBs is formed in lower yield with high LET radiation. T he complexity of DNA DSBs induced by HZE radiation will be discussed in terms of reduced repair efficiency and provide scope to model different sub-classes of DSBs as precursors that may lead to the detrimental health effects of HZE radiation.

  16. Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation

    Science.gov (United States)

    O'Neill, Peter; Harper, Jane; Anderson, Jennifer a.; Cucinnota, Francis A.

    2007-01-01

    -irradiation the rate of DSB repair is similar in the presence or absence vanillin, thus suggesting the repair of a sub-set of DSBs induced by low dose, low-LET radiation does not require DNA-PK(sub cs). This sub-set of DSBs is formed in lower yield with high LET radiation. T he complexity of DNA DSBs induced by HZE radiation will be discussed in terms of reduced repair efficiency and provide scope to model different sub-classes of DSBs as precursors that may lead to the detrimental health effects of HZE radiation.

  17. 53BP1 regulates DSB repair using Rif1 to control 5' end resection.

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    Zimmermann, Michal; Lottersberger, Francisca; Buonomo, Sara B; Sfeir, Agnel; de Lange, Titia

    2013-02-08

    The choice between double-strand break (DSB) repair by either homology-directed repair (HDR) or nonhomologous end joining (NHEJ) is tightly regulated. Defects in this regulation can induce genome instability and cancer. 53BP1 is critical for the control of DSB repair, promoting NHEJ, and inhibiting the 5' end resection needed for HDR. Using dysfunctional telomeres and genome-wide DSBs, we identify Rif1 as the main factor used by 53BP1 to impair 5' end resection. Rif1 inhibits resection involving CtIP, BLM, and Exo1; limits accumulation of BRCA1/BARD1 complexes at sites of DNA damage; and defines one of the mechanisms by which 53BP1 causes chromosomal abnormalities in Brca1-deficient cells. These data establish Rif1 as an important contributor to the control of DSB repair by 53BP1.

  18. Higher-order chromatin structure in DSB induction, repair and misrepair.

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    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

    2010-01-01

    Double-strand breaks (DSBs), continuously introduced into DNA by cell metabolism, ionizing radiation and some chemicals, are the biologically most deleterious type of genome damage, and must be accurately repaired to protect genomic integrity, ensure cell survival, and prevent carcinogenesis. Although a huge amount of information has been published on the molecular basis and biological significance of DSB repair, our understanding of DSB repair and its spatiotemporal arrangement is still incomplete. In particular, the role of higher-order chromatin structure in DSB induction and repair, movement of DSBs and the mechanism giving rise to chromatin exchanges, and many other currently disputed questions are discussed in this review. Finally, a model explaining the formation of chromosome translocations is proposed.

  19. Biochemical DSB-repair model for mammalian cells in G1 and early S phases of the cell cycle.

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    Taleei, Reza; Nikjoo, Hooshang

    2013-08-30

    The paper presents a model of double strand breaks (DSB) repair in G1 and early S phases of the cell cycle. The model is based on a plethora of published information on biochemical modification of DSB induced by ionizing radiation. So far, three main DSB repair pathways have been identified, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). During G1 and early S phases of the cell cycle, NHEJ and MMEJ repair pathways are activated dependent on the type of double strand breaks. Simple DSB are a substrate for NHEJ, while complex DSB and DSB in heterochromatin require further end processing. Repair of all DSB start with NHEJ presynaptic processes, and depending on the type of DSB pursue simple ligation, further end processing prior to ligation, or resection. Using law of mass action the model is translated into a mathematical formalism. The solution of the formalism provides the step by step and overall repair kinetics. The overall repair kinetics are compared with the published experimental measurements. Our calculations are in agreement with the experimental results and show that the complex types of DSBs are repaired with slow repair kinetics. The G1 and early S phase model could be employed to predict the kinetics of DSB repair for damage induced by high LET radiation.

  20. UbcH7 regulates 53BP1 stability and DSB repair.

    Science.gov (United States)

    Han, Xiangzi; Zhang, Lei; Chung, Jinsil; Mayca Pozo, Franklin; Tran, Amanda; Seachrist, Darcie D; Jacobberger, James W; Keri, Ruth A; Gilmore, Hannah; Zhang, Youwei

    2014-12-09

    DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.

  1. The power of DNA double-strand break (DSB) repair testing to predict breast cancer susceptibility.

    Science.gov (United States)

    Keimling, Marlen; Deniz, Miriam; Varga, Dominic; Stahl, Andreea; Schrezenmeier, Hubert; Kreienberg, Rolf; Hoffmann, Isabell; König, Jochem; Wiesmüller, Lisa

    2012-05-01

    Most presently known breast cancer susceptibility genes have been linked to DSB repair. To identify novel markers that may serve as indicators for breast cancer risk, we performed DSB repair analyses using a case-control design. Thus, we examined 35 women with defined familial history of breast and/or ovarian cancer (first case group), 175 patients with breast cancer (second case group), and 245 healthy women without previous cancer or family history of breast cancer (control group). We analyzed DSB repair in peripheral blood lymphocytes (PBLs) by a GFP-based test system using 3 pathway-specific substrates. We found increases of microhomology-mediated nonhomologous end joining (mmNHEJ) and nonconservative single-strand annealing (SSA) in women with familial risk vs. controls (P=0.0001-0.0022) and patients with breast cancer vs. controls (P=0.0004-0.0042). Young age (DSB repair activities in PBLs as method to estimate breast cancer susceptibility beyond limitations of genotyping and to predict responsiveness to therapeutics targeting DSB repair-dysfunctional tumors.

  2. Human transcriptional coactivator PC4 stimulates DNA end joining and activates DSB repair activity.

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    Batta, Kiran; Yokokawa, Masatoshi; Takeyasu, Kunio; Kundu, Tapas K

    2009-01-23

    Human transcriptional coactivator PC4 is a highly abundant nuclear protein that is involved in diverse cellular processes ranging from transcription to chromatin organization. Earlier, we have shown that PC4, a positive activator of p53, overexpresses upon genotoxic insult in a p53-dependent manner. In the present study, we show that PC4 stimulates ligase-mediated DNA end joining irrespective of the source of DNA ligase. Pull-down assays reveal that PC4 helps in the association of DNA ends through its C-terminal domain. In vitro nonhomologous end-joining assays with cell-free extracts show that PC4 enhances the joining of noncomplementary DNA ends. Interestingly, we found that PC4 activates double-strand break (DSB) repair activity through stimulation of DSB rejoining in vivo. Together, these findings demonstrate PC4 as an activator of nonhomologous end joining and DSB repair activity.

  3. Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

    Science.gov (United States)

    Seluanov, Andrei; Mao, Zhiyong; Gorbunova, Vera

    2010-01-01

    DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency. PMID:20864925

  4. Analysis of DNA double-strand break (DSB) repair in mammalian cells.

    Science.gov (United States)

    Seluanov, Andrei; Mao, Zhiyong; Gorbunova, Vera

    2010-09-08

    DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

  5. Repair of I-SceI induced DSB at a specific site of chromosome in human cells: influence of low-dose, low-dose-rate gamma-rays.

    Science.gov (United States)

    Yatagai, Fumio; Suzuki, Masao; Ishioka, Noriaki; Ohmori, Hitoshi; Honma, Masamitsu

    2008-11-01

    We investigated the influence of low-dose, low-dose-rate gamma-ray irradiation on DNA double strand break (DSB) repair in human lymphoblastoid TK6 cells. A single DSB was introduced at intron 4 of the TK+ allele (chromosome 17) by transfection with the I-SceI expression vector pCBASce. We assessed for DSB repair due to non-homologous end-joining (NHEJ) by determining the generation of TK-deficient mutants in the TK6 derivative TSCE5 (TK +/-) carrying an I-SceI recognition site. We similarly estimated DSB repair via homologous recombination (HR) at the same site in the derived compound heterozygote (TK-/-) cell line TSCER2 that carries an additional point mutation in exon 5. The NHEJ repair of DSB was barely influenced by pre-irradiation of the cells with 30 mGy gamma-rays at 1.2 mGy h(-1). DSB repair by HR, in contrast, was enhanced by approximately 50% after pre-irradiation of the cells under these conditions. Furthermore, when I-SceI digestion was followed by irradiation at a dose of 8.5 mGy, delivered at a dose rate of only 0.125 mGy h(-1), HR repair efficiency was enhanced by approximately 80%. This experimental approach can be applied to characterize DSB repair in the low-dose region of ionizing radiation.

  6. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  7. Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

    Science.gov (United States)

    Smith, Johanna A; Wang, Feng-Xiang; Zhang, Hui; Wu, Kou-Juey; Williams, Kevin Jon; Daniel, René

    2008-01-22

    Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

  8. An RNA Polymerase II-coupled function for histone H3K36 methylation in checkpoint activation and DSB repair

    OpenAIRE

    Jha, Deepak Kumar; Brian D Strahl

    2014-01-01

    Histone modifications are major determinants of DNA double-strand break (DSB) response and repair. Here we elucidate a DSB repair function for transcription-coupled Set2 methylation at H3 lysine 36 (H3K36me). Cells devoid of Set2/H3K36me are hypersensitive to DNA-damaging agents and site-specific DSBs, fail to properly activate the DNA-damage checkpoint, and show genetic interactions with DSB-sensing and repair machinery. Set2/H3K36me3 is enriched at DSBs, and loss of Set2 results in altered ...

  9. Importance of the cell cycle phase for the choice of the appropriate DSB repair pathway, for genome stability maintenance: the trans-S double-strand break repair model.

    Science.gov (United States)

    Delacôte, Fabien; Lopez, Bernard S

    2008-01-01

    A DNA double-strand break (DSB) is a highly harmful lesion that can lead to genome rearrangements. Two main pathways compete for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). Depending on the cell cycle phase, the choice of one DSB repair pathway over the other will secure genome stability maintenance or in contrast will increase the risk of genetic instability. HR with the sister chromatid is an efficient way to maintain genome stability, for damage occurring at a post-replication stage. However, in G(1) checkpoint-defective cells, DSBs produced in the G(1) phase and not repaired by NHEJ, can progress through S phase and be processed by HR in late S/G(2) phase. We propose the "trans-S DSB repair" model to account for these data. In this situation HR cannot use the sister chromatid (which is also broken at the same locus) and is thus forced to use ectopic homologous sequences dispersed through the genome, increasing the risk of genetic instability. This shows that the two DSB repair pathways can compete through the cell cycle and underlines the importance of the association between the cell cycle checkpoint and the appropriate DNA repair pathway for genome stability maintenance.

  10. ATM prevents DSB formation by coordinating SSB repair and cell cycle progression.

    Science.gov (United States)

    Khoronenkova, Svetlana V; Dianov, Grigory L

    2015-03-31

    DNA single-strand breaks (SSBs) arise as a consequence of spontaneous DNA instability and are also formed as DNA repair intermediates. Their repair is critical because they otherwise terminate gene transcription and generate toxic DNA double-strand breaks (DSBs) on replication. To prevent the formation of DSBs, SSB repair must be completed before DNA replication. To accomplish this, cells should be able to detect unrepaired SSBs, and then delay cell cycle progression to allow more time for repair; however, to date there is no evidence supporting the coordination of SSB repair and replication in human cells. Here we report that ataxia-telangiectasia mutated kinase (ATM) plays a major role in restricting the replication of SSB-containing DNA and thus prevents DSB formation. We show that ATM is activated by SSBs and coordinates their repair with DNA replication. SSB-mediated ATM activation is followed by a G1 cell cycle delay that allows more time for repair and thus prevents the replication of damaged DNA and DSB accrual. These findings establish an unanticipated role for ATM in the signaling of DNA SSBs and provide important insight into the molecular defects leading to genetic instability in patients with ataxia-telangiectasia.

  11. Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

    Science.gov (United States)

    Lee, Kyung-Jong; Saha, Janapriya; Sun, Jingxin; Fattah, Kazi R; Wang, Shu-Chi; Jakob, Burkhard; Chi, Linfeng; Wang, Shih-Ya; Taucher-Scholz, Gisela; Davis, Anthony J; Chen, David J

    2016-02-29

    Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

  12. An RNA polymerase II-coupled function for histone H3K36 methylation in checkpoint activation and DSB repair.

    Science.gov (United States)

    Jha, Deepak Kumar; Strahl, Brian D

    2014-06-09

    Histone modifications are major determinants of DNA double-strand break (DSB) response and repair. Here we elucidate a DSB repair function for transcription-coupled Set2 methylation at H3 lysine 36 (H3K36me). Cells devoid of Set2/H3K36me are hypersensitive to DNA-damaging agents and site-specific DSBs, fail to properly activate the DNA-damage checkpoint, and show genetic interactions with DSB-sensing and repair machinery. Set2/H3K36me3 is enriched at DSBs, and loss of Set2 results in altered chromatin architecture and inappropriate resection during G1 near break sites. Surprisingly, Set2 and RNA polymerase II are programmed for destruction after DSBs in a temporal manner--resulting in H3K36me3 to H3K36me2 transition that may be linked to DSB repair. Finally, we show a requirement of Set2 in DSB repair in transcription units--thus underscoring the importance of transcription-dependent H3K36me in DSB repair.

  13. DSB repair model for mammalian cells in early S and G1 phases of the cell cycle: application to damage induced by ionizing radiation of different quality.

    Science.gov (United States)

    Taleei, Reza; Girard, Peter M; Nikjoo, Hooshang

    2015-02-01

    The purpose of this work is to test the hypothesis that kinetics of double strand breaks (DSB) repair is governed by complexity of DSB. To test the hypothesis we used our recent published mechanistic mathematical model of DSB repair for DSB induced by selected protons, deuterons, and helium ions of different energies representing radiations of different qualities. In light of recent advances in experimental and computational techniques, the most appropriate method to study cellular responses in radiation therapy, and exposures to low doses of ionizing radiations is using mechanistic approaches. To this end, we proposed a 'bottom-up' approach to study cellular response that starts with the DNA damage. Monte Carlo track structure method was employed to simulate initial damage induced in the genomic DNA by direct and indirect effects. Among the different types of DNA damage, DSB are known to be induced in simple and complex forms. The DSB repair model in G1 and early S phases of the cell cycle was employed to calculate the repair kinetics. The model considers the repair of simple and complex DSB, and the DSB produced in the heterochromatin. The inverse sampling method was used to calculate the repair kinetics for each individual DSB. The overall repair kinetics for 500 DSB induced by single tracks of the radiation under test were compared with experimental results. The results show that the model is capable of predicting the repair kinetics for the DSB induced by radiations of different qualities within an accepted range of uncertainty.

  14. 53BP1 and the LINC Complex Promote Microtubule-Dependent DSB Mobility and DNA Repair.

    Science.gov (United States)

    Lottersberger, Francisca; Karssemeijer, Roos Anna; Dimitrova, Nadya; de Lange, Titia

    2015-11-05

    Increased mobility of chromatin surrounding double-strand breaks (DSBs) has been noted in yeast and mammalian cells but the underlying mechanism and its contribution to DSB repair remain unclear. Here, we use a telomere-based system to track DNA damage foci with high resolution in living cells. We find that the greater mobility of damaged chromatin requires 53BP1, SUN1/2 in the linker of the nucleoskeleton, and cytoskeleton (LINC) complex and dynamic microtubules. The data further demonstrate that the excursions promote non-homologous end joining of dysfunctional telomeres and implicated Nesprin-4 and kinesins in telomere fusion. 53BP1/LINC/microtubule-dependent mobility is also evident at irradiation-induced DSBs and contributes to the mis-rejoining of drug-induced DSBs in BRCA1-deficient cells showing that DSB mobility can be detrimental in cells with numerous DSBs. In contrast, under physiological conditions where cells have only one or a few lesions, DSB mobility is proposed to prevent errors in DNA repair.

  15. MutS homologue hMSH5: recombinational DSB repair and non-synonymous polymorphic variants.

    Science.gov (United States)

    Wu, Xiling; Xu, Yang; Feng, Katey; Tompkins, Joshua D; Her, Chengtao

    2013-01-01

    Double-strand breaks (DSBs) constitute the most deleterious form of DNA lesions that can lead to genome alterations and cell death, and the vast majority of DSBs arise pathologically in response to DNA damaging agents such as ionizing radiation (IR) and chemotherapeutic agents. Recent studies have implicated a role for the human MutS homologue hMSH5 in homologous recombination (HR)-mediated DSB repair and the DNA damage response. In the present study, we show that hMSH5 promotes HR-based DSB repair, and this property resides in the carboxyl-terminal portion of the protein. Our results demonstrate that DSB-triggered hMSH5 chromatin association peaks at the proximal regions of the DSB and decreases gradually with increased distance from the break. Furthermore, the DSB-triggered hMSH5 chromatin association is preceded by and relies on the assembly of hMRE11 and hRad51 at the proximal regions of the DSB. Lastly, the potential effects of hMSH5 non-synonymous variants (L85F, Y202C, V206F, R351G, L377F, and P786S) on HR and cell survival in response to DSB-inducing anticancer agents have been analyzed. These experiments show that the expression of hMSH5 variants elicits different survival responses to anticancer drugs cisplatin, bleomycin, doxorubicin and camptothecin. However, the effects of hMSH5 variants on survival responses to DSB-inducing agents are not directly correlated to their effects exerted on HR-mediated DSB repair, suggesting that the roles of hMSH5 variants in the processes of DNA damage response and repair are multifaceted.

  16. A sensitive test for the detection of specific DSB repair defects in primary cells from breast cancer specimens.

    Science.gov (United States)

    Keimling, Marlen; Kaur, Jatinder; Bagadi, Sarangadhara Appala Raju; Kreienberg, Rolf; Wiesmüller, Lisa; Ralhan, Ranju

    2008-08-01

    Increasing evidence indicates that breast cancer pathogenesis is linked with DNA double-strand break (DSB) repair dysfunction. This conclusion is based on advances in the study of functions of breast cancer susceptibility genes such as BRCA1 and BRCA2, on the identification of breast cancer-associated changes regarding the genetics, expression, and localization of multiple DSB repair factors, and on observations indicating enhanced radiation-induced chromosomal damage in cells from predisposed individuals and sporadic breast cancer patients. In this pilot study, we describe a sensitive method for the analysis of DSB repair functions in mammary carcinomas. Using this method we firstly document alterations in pathway-specific DSB repair activities in primary cells originating from familial as well as sporadic breast cancer. In particular, we identified increases in the mutagenic nonhomologous end joining and single-strand annealing mechanisms in sporadic breast cancers with wild-type BRCA1 and BRCA2, and, thus, similar phenotypes to tumors with mutant alleles of BRCA1 and BRCA2. This suggests that detection of error-prone DSB repair activities may be useful to extend the limits of genotypic characterization of high-risk susceptibility genes. This method may, therefore, serve as a marker for breast cancer risk assessment and, even more importantly, for the prediction of responsiveness to targeted therapies such as to inhibitors of poly(ADP-ribose)polymerase (PARP1).

  17. Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells

    Science.gov (United States)

    Pan, Hongjie; Zhang, Xuan; Jiang, Hanwei; Jiang, Xiaohua; Wang, Liu; Qi, Qi; Bi, Yuan; Wang, Jian; Shi, Qinghua; Li, Runsheng

    2017-01-01

    The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3+/− germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse. PMID:28290521

  18. Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells.

    Science.gov (United States)

    Pan, Hongjie; Zhang, Xuan; Jiang, Hanwei; Jiang, Xiaohua; Wang, Liu; Qi, Qi; Bi, Yuan; Wang, Jian; Shi, Qinghua; Li, Runsheng

    2017-03-14

    The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3(+/-) germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse.

  19. Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

    Science.gov (United States)

    Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

    2007-01-01

    We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

  20. Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors.

    Science.gov (United States)

    Goglia, Alexander G; Delsite, Robert; Luz, Antonio N; Shahbazian, David; Salem, Ahmed F; Sundaram, Ranjini K; Chiaravalli, Jeanne; Hendrikx, Petrus J; Wilshire, Jennifer A; Jasin, Maria; Kluger, Harriet M; Glickman, J Fraser; Powell, Simon N; Bindra, Ranjit S

    2015-02-01

    Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA-damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radiosensitization. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. Here, we report the design and execution of a high-throughput, cell-based small molecule screen for novel DSB repair inhibitors. We miniaturized our recently developed dual NHEJ and HR reporter system into a 384-well plate-based format and interrogated a diverse library of 20,000 compounds for molecules that selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits that potently inhibit DSB repair, and we have validated their functional activity in a comprehensive panel of orthogonal secondary assays. A selection of these inhibitors was found to radiosensitize cancer cell lines in vitro, which suggests that they may be useful as novel chemo- and radio sensitizers. Surprisingly, we identified several FDA-approved drugs, including the calcium channel blocker mibefradil dihydrochloride, that demonstrated activity as DSB repair inhibitors and radiosensitizers. These findings suggest the possibility for repurposing them as tumor cell radiosensitizers in the future. Accordingly, we recently initiated a phase I clinical trial testing mibefradil as a glioma radiosensitizer.

  1. Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair.

    Science.gov (United States)

    Gouge, Jérôme; Rosario, Sandrine; Romain, Félix; Poitevin, Frédéric; Béguin, Pierre; Delarue, Marc

    2015-04-15

    Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3'-protruding ends of a DNA double-strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non-homologous end-joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro-homology (MH) base pair and one nascent base pair. This structure reveals how the N-terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site-directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.

  2. Participation of DNA-PKcs in DSB repair after exposure to high- and low-LET radiation.

    Science.gov (United States)

    Anderson, Jennifer A; Harper, Jane V; Cucinotta, Francis A; O'Neill, Peter

    2010-08-01

    Cellular lesions (e.g. DSBs) are induced into DNA upon exposure to radiation, with DSB complexity increasing with radiation ionization density. Using M059K and M059J human glioblastoma cells (proficient and deficient in DNA-PKcs activity, respectively), we investigated the repair of DNA damage, including DSBs, induced by high- and low-LET radiation [gamma rays, alpha particles and high-charge and energy (HZE) ions]. In the absence of DNA-PKcs activity, less DSB repair and increased recruitment of RAD51 was seen at 24 h. After exposure to (56)Fe heavy ions, the number of cells with RAD51 tracks was less than the number of cells with gamma-H2AX at 24 h with both cell lines. Using alpha particles, comparable numbers of cells with visible gamma-H2AX and RAD51 were seen at 24 h in both cell lines. M059J cells irradiated with alpha particles accumulated in S phase, with a greater number of cyclin A and RAD51 co-stained cells seen at 24 h compared with M059K cells, where an S-phase block is absent. It is proposed that DNA-PKcs plays a role in the repair of some frank DSBs, which are longer-lived in NHEJ-deficient cells, and some non-DSB clustered damage sites that are converted into DSBs at replication as the cell cycles through to S phase.

  3. High throughput measurement of γH2AX DSB repair kinetics in a healthy human population.

    Science.gov (United States)

    Sharma, Preety M; Ponnaiya, Brian; Taveras, Maria; Shuryak, Igor; Turner, Helen; Brenner, David J

    2015-01-01

    The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.

  4. Topoisomerase degradation, DSB repair, p53 and IAPs in cancer cell resistance to camptothecin-like topoisomerase I inhibitors.

    Science.gov (United States)

    Tomicic, Maja T; Kaina, Bernd

    2013-01-01

    Topoisomerase I (TOP1) inhibitors applied in cancer therapy such as topotecan and irinotecan are derivatives of the natural alkaloid camptothecin (CPT). The mechanism of CPT poisoning of TOP1 rests on inhibition of the re-ligation function of the enzyme resulting in the stabilization of the TOP1-cleavable complex. In the presence of CPTs this enzyme-DNA complex impairs transcription and DNA replication, resulting in fork stalling and the formation of DNA double-strand breaks (DSB) in proliferating cells. As with most chemotherapeutics, intrinsic and acquired drug resistance represents a hurdle that limits the success of CPT therapy. Preclinical data indicate that resistance to CPT-based drugs might be caused by factors such as (a) poor drug accumulation in the tumor, (b) high rate of drug efflux, (c) mutations in TOP1 leading to failure in CPT docking, or (d) altered signaling triggered by the drug-TOP1-DNA complex, (e) expression of DNA repair proteins, and (f) failure to activate cell death pathways. This review will focus on the issues (d-f). We discuss degradation of TOP1 as part of the repair pathway in the processing of TOP1 associated DNA damage, give a summary of proteins involved in repair of CPT-induced replication mediated DSB, and highlight the role of p53 and inhibitors of apoptosis proteins (IAPs), particularly XIAP and survivin, in cancer cell resistance to CPT-like chemotherapeutics.

  5. Artemis is required to improve the accuracy of repair of double-strand breaks with 5'-blocked termini generated from non-DSB-clustered lesions.

    Science.gov (United States)

    Malyarchuk, Svitlana; Castore, Reneau; Shi, Runhua; Harrison, Lynn

    2013-05-01

    Clustered DNA lesions are defined as ≥2 damage events within 20 bp. Oxidised bases, abasic (AP) sites, single-strand breaks and double-strand breaks (DSBs) exist in radiation-induced clusters, and these lesions are more difficult to repair and can be more mutagenic than single lesions. Understanding clustered lesion repair is therefore important for the design of complementary treatments to enhance radiotherapy. Non-DSB-clustered lesions consisting of opposing AP sites can be converted to DSBs by base excision repair, and non-homologous end-joining (NHEJ) plays a role in repairing these DSBs. Artemis is an endonuclease that removes blocking groups from DSB termini during NHEJ. Hence, we hypothesised that Artemis plays a role in the processing of DSBs or complex DSBs generated from non-DSB-clustered lesions. We examined the repair of clusters containing two or three lesions in wild-type (WT) or Artemis-deficient (ART(-/-)) mouse fibroblasts using a reporter plasmid. Each cluster contained two opposing tetrahydrofurans (an AP site analogue), which AP endonuclease can convert to a DSB with blocked 5' termini. Loss of Artemis did not decrease plasmid survival, but did result in more mutagenic repair with plasmids containing larger deletions. This increase in deletions did not occur with ClaI-linearised plasmid. Since Mre11 has been implicated in deletional NHEJ, we used small interfering RNA to reduce Mre11 in WT and ART(-/-) cells, but decreasing Mre11 did not change the size of deletions in the repair products. This work implicates Artemis in limiting the deletions introduced during repair of 5'-blocked termini DSBs generated from non-DSB-clustered lesions. Decreasing repair accuracy without decreasing repair capacity could result in mutated cells surviving irradiation. Inhibiting Artemis in normal cells could promote carcinogenesis, while in tumour cells enhanced mutagenic repair following irradiation could promote tumour recurrence.

  6. Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Constantinescu, Dan [Department of Cell Biology-Physiology, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Pittsburgh Development Center, Magee-Women' s Research Institute, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Csoka, Antonei B. [Division of Geriatrics, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA 15260 (United States); Navara, Christopher S. [Division of Developmental and Regenerative Medicine, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Pittsburgh Development Center, Magee-Women' s Research Institute, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Schatten, Gerald P., E-mail: schattengp@upmc.edu [Division of Developmental and Regenerative Medicine, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Department of Cell Biology-Physiology, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Pittsburgh Development Center, Magee-Women' s Research Institute, University of Pittsburgh, Pittsburgh, PA 15260 (United States)

    2010-10-15

    Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.

  7. Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts.

    Science.gov (United States)

    Constantinescu, Dan; Csoka, Antonei B; Navara, Christopher S; Schatten, Gerald P

    2010-10-15

    Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.

  8. The C. elegans DSB-2 protein reveals a regulatory network that controls competence for meiotic DSB formation and promotes crossover assurance.

    Science.gov (United States)

    Rosu, Simona; Zawadzki, Karl A; Stamper, Ericca L; Libuda, Diana E; Reese, Angela L; Dernburg, Abby F; Villeneuve, Anne M

    2013-01-01

    For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1) ensures that sufficient DSBs are made to guarantee CO formation and (2) prevents excessive DSB levels that could have deleterious

  9. A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors.

    Science.gov (United States)

    Tilgner, K; Neganova, I; Moreno-Gimeno, I; Al-Aama, J Y; Burks, D; Yung, S; Singhapol, C; Saretzki, G; Evans, J; Gorbunova, V; Gennery, A; Przyborski, S; Stojkovic, M; Armstrong, L; Jeggo, P; Lako, M

    2013-08-01

    DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final 'end-joining' step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.

  10. High throughput measurement of γH2AX DSB repair kinetics in a healthy human population.

    Directory of Open Access Journals (Sweden)

    Preety M Sharma

    Full Text Available The Columbia University RABiT (Rapid Automated Biodosimetry Tool quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY (r=0.257, P=0.02 and a negative correlation with residuals (r=-0.521, P=<0.0001. A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001. Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.

  11. Transcriptional elongation factor ENL phosphorylated by ATM recruits polycomb and switches off transcription for DSB repair.

    Science.gov (United States)

    Ui, Ayako; Nagaura, Yuko; Yasui, Akira

    2015-05-07

    Transcription is repressed if a DNA double-strand break (DSB) is introduced in close proximity to a transcriptional activation site at least in part by H2A-ubiquitination. While ATM signaling is involved, how it controls H2A-ubiquitination remains unclear. Here, we identify that, in response to DSBs, a transcriptional elongation factor, ENL (MLLT1), is phosphorylated by ATM at conserved SQ sites. This phosphorylation increases the interaction between ENL and the E3-ubiquitin-ligase complex of Polycomb Repressive Complex 1 (PRC1) via BMI1. This interaction promotes enrichment of PRC1 at transcription elongation sites near DSBs to ubiquitinate H2A leading to transcriptional repression. ENL SQ sites and BMI1 are necessary for KU70 accumulation at DSBs near active transcription sites and cellular resistance to DSBs. Our data suggest that ATM-dependent phosphorylation of ENL functions as switch from elongation to Polycomb-mediated repression to preserve genome integrity.

  12. Genistein sensitizes sarcoma cells in vitro and in vivo by enhancing apoptosis and by inhibiting DSB repair pathways.

    Science.gov (United States)

    Liu, X X; Sun, C; Jin, X D; Li, P; Zheng, X G; Zhao, T; Li, Q

    2016-06-01

    The aim of this work was to investigate the radiosensitization effects of genistein on mice sarcoma cells and the corresponding biological mechanisms in vitro and in vivo Using the non-toxic dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at 50% cell survival (IC50) was 1.45 for S180 cells. For mice cotreated with genistein and X-rays, the excised tumor tissues had reduced blood vessels and decreased size and volume compared with the control and irradiation-only groups. Moreover, a significant increase in apoptosis was accompanied by upregulation of Bax and downregulation of Bcl-2 in the mitochondria, and lots of cytochrome c being transferred to the cytoplasm. Furthermore, X-rays combined with genistein inhibited the activity of DNA-PKcs, so DNA-injured sites were dominated by Ku70/80, leading to incompleteness of homologous recombination (HR) and non-homologous end-joining (NHEJ) repairs and the eventual occurrence of cell apoptosis. Our study, for the first time, demonstrated that genistein sensitized sarcoma cells to X-rays and that this radiosensitizing effect depended on induction of the mitochondrial apoptosis pathway and inhibition of the double-strand break (DSB) repair pathways.

  13. In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

    DEFF Research Database (Denmark)

    Kasten-Pisula, Ulla; Vronskaja, Svetlana; Overgaard, Jens

    2008-01-01

    BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins....... MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively...... in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity. Udgivelsesdato: 2008-Mar...

  14. TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair.

    Science.gov (United States)

    Gazy, Inbal; Zeevi, David A; Renbaum, Paul; Zeligson, Sharon; Eini, Lital; Bashari, Dana; Smith, Yoav; Lahad, Amnon; Goldberg, Michal; Ginsberg, Doron; Levy-Lahad, Ephrat

    2015-01-01

    Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, pDSB repair in malignancy. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, pDSB repair in malignancy.

  15. KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair.

    Science.gov (United States)

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2011-10-01

    Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Identification and Analysis of MS5(d): A Gene That Affects Double-Strand Break (DSB) Repair during Meiosis I in Brassica napus Microsporocytes.

    Science.gov (United States)

    Zeng, Xinhua; Yan, Xiaohong; Yuan, Rong; Li, Keqi; Wu, Yuhua; Liu, Fang; Luo, Junling; Li, Jun; Wu, Gang

    2016-01-01

    Here, we report the identification of the Brassica-specific gene MS5(d), which is responsible for male sterility in Brassica napus. The MS5(d) gene is highly expressed in the microsporocyte and encodes a protein that localizes to the nucleus. Light microscopy analyses have demonstrated that the MS5(d) gene affects microsporocyte meiosis in the thermosensitive genic male sterility line TE5A. Sequence comparisons and genetic complementation revealed a C-to-T transition in MS5(d), encoding a Leu-to-Phe (L281F) substitution and causing abnormal male meiosis in TE5A. These findings suggest arrested meiotic chromosome dynamics at pachytene. Furthermore, immunofluorescence analyses showed that double-strand break (DSB) formation and axial elements were normal but that DSB repair and spindle behavior were aberrant in TE5A meiocytes. Collectively, our results indicate that MS5(d) likely encodes a protein required for chromosomal DSB repair at early stages of meiosis in B. napus.

  17. Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Darroudi, Firouz [Department of Toxicogenetics, Leiden University Medical Centre, Einthovenweg 20, 2300RC Leiden (Netherlands)]. E-mail: F.Darroudi@LUMC.NL; Wiegant, Wouter [Department of Toxicogenetics, Leiden University Medical Centre, Einthovenweg 20, 2300RC Leiden (Netherlands); Meijers, Matty [Department of Toxicogenetics, Leiden University Medical Centre, Einthovenweg 20, 2300RC Leiden (Netherlands); Friedl, Anna A. [Radiobiological Institute, University of Munich, Munich (Germany); Institute of Radiobiology, GSF National Research Center for Environment and Health, Neuherberg (Germany); Burg, Mirjam van der [Department of Immunology, Erasmus Medical Centre, Rotterdam (Netherlands); Fomina, Janna [Department of Toxicogenetics, Leiden University Medical Centre, Einthovenweg 20, 2300RC Leiden (Netherlands); Dongen, Jacques J.M. van [Department of Immunology, Erasmus Medical Centre, Rotterdam (Netherlands); Gent, Dik C. van [Department of Cell Biology and Genetics, Erasmus Medical Centre, Rotterdam (Netherlands); Zdzienicka, Malgorzata Z. [Department of Toxicogenetics, Leiden University Medical Centre, Einthovenweg 20, 2300RC Leiden (Netherlands); Department of Molecular Cell Genetics, Collegium Medicum, N. Corpernicus University, Bydgoszcz (Poland)

    2007-02-03

    We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2-3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and {gamma}H2AX foci formation after ionizing irradiation, indicate that 15-20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G{sub 1}/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2-4-fold higher compared to normal fibroblasts. Irradiation in G{sub 2} resulted in a higher frequency of chromatid-type aberrations (1.5-3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G{sub 1} cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G{sub 1}-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4 {>=} AT > Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when

  18. Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle.

    Science.gov (United States)

    Darroudi, Firouz; Wiegant, Wouter; Meijers, Matty; Friedl, Anna A; van der Burg, Mirjam; Fomina, Janna; van Dongen, Jacques J M; van Gent, Dik C; Zdzienicka, Małgorzata Z

    2007-02-03

    We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2-3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and gammaH2AX foci formation after ionizing irradiation, indicate that 15-20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G(1)/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2-4-fold higher compared to normal fibroblasts. Irradiation in G(2) resulted in a higher frequency of chromatid-type aberrations (1.5-3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G(1) cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G(1)-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4> or =AT>Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when cells are devoid of

  19. DNA双链断裂NHEJ修复及其与肿瘤的研究%Non-homologous end joining pathway of DSB repair and cancer

    Institute of Scientific and Technical Information of China (English)

    张耀伟

    2010-01-01

    非同源末端连接是哺乳动物最主要的DNA双链断裂(DSB)连接方式.肿瘤细胞非同源末端连接能力的提高与其放化疗抵抗有关,抑制肿瘤细胞非同源末端连接能力,可能增加其对放化疗的敏感性.因此,参与非同源末端连接的修复因子可能成为肿瘤分子靶向治疗及放化疗增敏的新治疗点.%Non-homologous end joining (NHEJ) is the major pathway for repairing DNA doublestrand break (DSB) in mammalian species. The capacity of NHEJ increases in tumor cell,which plays a role in radiation/chemotherapy-resistant agent Inhibiting DSB rejoining may play a crucial role in the enhancement of cellular radiation/chemotherapy-sensitizing. Thus, the protein molecule enrolled in NHEJ may be new potential targets for radiation/chemotherapy -sensitizing.

  20. Plant γH2AX foci are required for proper DNA DSB repair responses and colocalize with E2F factors.

    Science.gov (United States)

    Lang, Julien; Smetana, Ondrej; Sanchez-Calderon, Lenin; Lincker, Frédéric; Genestier, Julie; Schmit, Anne-Catherine; Houlné, Guy; Chabouté, Marie-Edith

    2012-04-01

    Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts to the phosphorylated histones H2AX (γH2AX) repair foci which are multiproteic nuclear complexes responsible for DSB sensing and signalling. However, neither the components of these foci nor their role are yet known in plants. In this paper, we describe the effects of γH2AX deficiency in Arabidopsis thaliana plants challenged with DSBs in terms of genotoxic sensitivity and E2F-mediated transcriptional responses. We further establish the existence, restrictive to the G1/S transition, of specific DSB-induced foci containing tobacco E2F transcription factors, in both A. thaliana roots and BY-2 tobacco cells. These E2F foci partially colocalize with γH2AX foci while their formation is ataxia telangiectasia mutated (ATM)-dependent, requires the E2F transactivation domain with its retinoblastoma-binding site and is optimal in the presence of functional H2AXs. Overall, our results unveil a new interplay between plant H2AX and E2F transcriptional activators during the DSB response.

  1. The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex.

    Science.gov (United States)

    Ge, Chunmin; Che, Lixiao; Du, Chunying

    2015-01-01

    BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.

  2. Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Satoshi Nakajima

    Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

  3. TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

    Science.gov (United States)

    Renbaum, Paul; Zeligson, Sharon; Eini, Lital; Bashari, Dana; Smith, Yoav; Lahad, Amnon; Goldberg, Michal; Ginsberg, Doron; Levy-Lahad, Ephrat

    2015-01-01

    Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RAD51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51

  4. More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny

    Directory of Open Access Journals (Sweden)

    Leyla Vahidi Ferdousi

    2014-11-01

    Full Text Available The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

  5. Impaired 53BP1/RIF1 DSB mediated end-protection stimulates CtIP-dependent end resection and switches the repair to PARP1-dependent end joining in G1.

    Science.gov (United States)

    Bakr, Ali; Köcher, Sabrina; Volquardsen, Jennifer; Petersen, Cordula; Borgmann, Kerstin; Dikomey, Ekkehard; Rothkamm, Kai; Mansour, Wael Y

    2016-09-06

    End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.

  6. RAG2 mutants alter DSB repair pathway choice in vivo and illuminate the nature of 'alternative NHEJ'.

    Science.gov (United States)

    Gigi, Vered; Lewis, Susanna; Shestova, Olga; Mijušković, Martina; Deriano, Ludovic; Meng, Wenzhao; Luning Prak, Eline T; Roth, David B

    2014-06-01

    DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

  7. The role of the Mre11-Rad50-Nbs1 complex in double-strand break repair-facts and myths.

    Science.gov (United States)

    Takeda, Shunichi; Hoa, Nguyen Ngoc; Sasanuma, Hiroyuki

    2016-08-01

    Homologous recombination (HR) initiates double-strand break (DSB) repair by digesting 5'-termini at DSBs, the biochemical reaction called DSB resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. Rad51 recombinase polymerizes along resected DNA, and the resulting Rad51-DNA complex undergoes homology search. Although DSB resection by the Mre11 nuclease plays a critical role in HR in Saccharomyces cerevisiae, it remains elusive whether DSB resection by Mre11 significantly contributes to HR-dependent DSB repair in mammalian cells. Depletion of Mre11 decreases the efficiency of DSB resection only by 2- to 3-fold in mammalian cells. We show that although Mre11 is required for efficient HR-dependent repair of ionizing-radiation-induced DSBs, Mre11 is largely dispensable for DSB resection in both chicken DT40 and human TK6 B cell lines. Moreover, a 2- to 3-fold decrease in DSB resection has virtually no impact on the efficiency of HR. Thus, although a large number of researchers have reported the vital role of Mre11-mediated DSB resection in HR, the role may not explain the very severe defect in HR in Mre11-deficient cells, including their lethality. We here show experimental evidence for the additional roles of Mre11 in (i) elimination of chemical adducts from DSB ends for subsequent DSB repair, and (ii) maintaining HR intermediates for their proper resolution.

  8. Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.

    Directory of Open Access Journals (Sweden)

    Hua Fung

    Full Text Available Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR or nonhomologous end-joining (NHEJ. For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny.

  9. SHP-1 overexpression increases the radioresistance of NPC cells by enhancing DSB repair, increasing S phase arrest and decreasing cell apoptosis.

    Science.gov (United States)

    Pan, Xiaofen; Mou, Jingjing; Liu, Sha; Sun, Ziyi; Meng, Rui; Zhou, Zhenwei; Wu, Gang; Peng, Gang

    2015-06-01

    The present study aimed to investigate the influence of SHP-1 on the radioresistance of the nasopharyngeal carcinoma (NPC) cell line CNE-2 and the relevant underlying mechanisms. The human NPC cell line CNE-2 was transfected with a lentivirus that contained the SHP-1 gene or a nonsense sequence (referred to as LP-H1802Lv201 and LP-NegLv201 cells, respectively). Cells were irradiated with different ionizing radiation (IR) doses. Cell survival, DNA double-strand breaks (DSBs), apoptosis, cell cycle distribution, and the expression of related proteins were assessed using colony formation assay, immunofluorescent assays (IFAs), flow cytometry (FCM) and western blot analyses, respectively. Compared with the control (CNE-2 cells) and LP-NegLv201 cells, LP-H1802Lv201 cells were more resistant to IR. IFAs showed that IR caused less histone H2AX phosphorylation (γH2AX) and RAD51 foci in the LP-H1802Lv201 cells. Compared with the control and LP-NegLv201 cells, LP-H1802Lv201 cells showed increased S phase arrest. After IR, the apoptotic rate of the LP-H1802Lv201 cells was lower in contrast to the control and LP-NegLv201 cells. Western blot analyses showed that IR increased the phosphorylation of ataxia telangiectasia mutated (ATM) kinase, checkpoint kinase 2 (CHK2), ataxia telangiectasia and Rad3-related (ATR) protein, checkpoint kinase 1 (CHK1) and p53. In LP-H1802Lv201 cells, the phosphorylation levels of ATM and CHK2 were significantly increased while the p53 phosphorylation level was decreased compared to these levels in the control and LP-NegLv201 cells. Phosphorylation of ATR and CHK1 did not show significant differences in the three cell groups. Overexpression of SHP-1 in the CNE-2 cells led to radioresistance and the radioresistance was related to enhanced DNA DSB repair, increased S phase arrest and decreased cell apoptosis.

  10. XRCC1 deficiency increased the DNA damage induced by γ-ray in HepG2 cell: Involvement of DSB repair and cell cycle arrest.

    Science.gov (United States)

    Niu, Yujie; Zhang, Xing; Zheng, Yuxin; Zhang, Rong

    2013-09-01

    γ-ray irradiation can induce DNA damages which include base damages, single-strand breaks and double-strand breaks in various type cells. The DNA repair protein XRCC1, as a part of the BER pathway, forms complexes with DNA polymerase beta, DNA ligase III and poly-ADP-ribose polymerase (PARP) in the repair of DNA single strand breaks and also affects the repair of double strand breaks. However, it is still not known well whether XRCC1 contributes to affect the irradiation sensitivity and DNA damage in HepG2 cell and the potential mechanism. Hence, the purpose of this study was to explore whether abrogation of XRCC1 gene expression by shRNA could reduce DNA repair and thus sensitize HepG2 cells to γ-ray. Cell viability was measured by Trypan blue staining and cloning efficiency assay. The DNA damage was detected by Comet assay. Apoptosis and cell cycle were detected by flow cytometry. The DNA-PKcs and gadd153 mRNA expression were determined by Real-time PCR. Our results showed that abrogation of XRCC 1 could sensitize HepG2 cells to γ-ray. This enhanced sensitivity could be attributed to the increased DNA damage and increased cell cycle arrest, which might be related with the increasing of DNA-PKcs and gadd153 mRNA expression. Therefore, our results suggested that the γ-ray irradiation sensitivity could be increased by targeting inhibition of XRCC1 in HepG2 cell.

  11. The efficiency of homologous recombination and non-homologous end joining systems in repairing double-strand breaks during cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Leonardo Bee

    Full Text Available This study investigated the efficiency of Non-Homologous End Joining (NHEJ and Homologous Recombination (HR repair systems in rejoining DNA double-strand breaks (DSB induced in CCD-34Lu cells by different γ-ray doses. The kinetics of DNA repair was assessed by analyzing the fluorescence decrease of γ-H2AX foci measured by SOID (Sum Of Integrated Density parameter and counting foci number in the time-interval 0.5-24 hours after irradiation. Comparison of the two methods showed that the SOID parameter was useful in determining the amount and the persistence of DNA damage signal after exposure to high or low doses of ionizing radiation. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1, S, and G2 phase cells on the basis of nuclear fluorescence of the CENP-F protein. Six hours after irradiation, γ-H2AX foci resolution was higher in G2 compared to G1 cells in which both NHEJ and HR can cooperate. The rejoining of γ-H2AX foci in G2 phase cells was, moreover, decreased by RI-1, the chemical inhibitor of HR, demonstrating that homologous recombination is at work early after irradiation. The relevance of HR in DSB repair was assessed in DNA-PK-deficient M059J cells and in CCD-34Lu treated with the DNA-PKcs inhibitor, NU7026. In both conditions, the kinetics of γ-H2AX demonstrated that DSBs repair was markedly affected when NHEJ was absent or impaired, even in G2 phase cells in which HR should be at work. The recruitment of RAD51 at DSB sites was, moreover, delayed in M059J and in NU7026 treated-CCD-34Lu, with respect to DNA-PKcs proficient cells and continued for 24 hours despite the decrease in DNA repair. The impairment of NHEJ affected the efficiency of the HR system and significantly decreased cell survival after ionizing radiation, confirming that DSB rejoining is strictly dependent on the integrity of the NHEJ repair system.

  12. 组蛋白去乙酰化酶抑制剂对DNA双链断裂修复路径的作用%Effect of Histone Deacetylase Inhibitors on DNA DSB Repair

    Institute of Scientific and Technical Information of China (English)

    孙有湘; 周克元; 李莉萍

    2013-01-01

    DNA双链断裂(double strand breaks,DSBs)对细胞生存是致命的.细胞内非同源末端连接(NHEJ)、重组修复(HDR)、单链退火修复(SSA)和微同源序列末端连接(MMEJ)等通路可竞争性修复DNA双链断裂损伤.在肿瘤细胞DNA中制造难以修复的基因损伤,诱导肿瘤细胞周期中止、坏死和凋亡是临床放、化疗的主要策略.组蛋白去乙酰化酶(histone deacetylase)作为抗肿瘤治疗的新靶标,其抑制剂(histone deacetylase inhibitors,HDACi)可显著降低肿瘤细胞DSBs修复能力,增强肿瘤细胞的放、化疗敏感性.研究显示,HDACi抑制了肿瘤细胞中具有正确修复倾向的HDR和经典NHEJ通路,具有错误修复倾向的SSA和MMEJ路径也可能牵涉其中.目前,HDACi作用于DSBs修复通路的分子机制已取得较大进展,但仍有许多问题有待阐明.%DNA double strand break (DSB) can be fatal to cells.The non-homologous end joining (NHEJ),homologous direct recombination (HDR),single strand annealing (SSA) and micro-homology mediated end joining (MMEJ) pathways are able to repair DSBs in vivo.A main strategy of clinical radiotherapy and chemotherapy is to induce chromosomal DNA damage that are difficult to repair,thus to cause cell cycle arrest,apoptosis or necrosis in tumor cells.As a new target of anti-tumor medicine,histonc deacetylase inhibitor (HDACi) effectively suppresses DSBs repair capacity and sensitize tumor cells to radiotherapy or chemotherapy.Recent research suggested that the error-free DSBs repair pathways HDR and classical-NHEJ were suppressed by HDACi in tumor cells,and the error-prone SSA and MMEJ pathways might also involved in.Although much progress have acquired on the mechanism of how HDACi affecting DSB repair pathways,there are still many questions need to be elucidated.

  13. TU-EF-304-10: Efficient Multiscale Simulation of the Proton Relative Biological Effectiveness (RBE) for DNA Double Strand Break (DSB) Induction and Bio-Effective Dose in the FLUKA Monte Carlo Radiation Transport Code

    Energy Technology Data Exchange (ETDEWEB)

    Moskvin, V; Tsiamas, P; Axente, M; Farr, J [St. Jude Children’s Research Hospital, Memphis, TN (United States); Stewart, R [University of Washington, Seattle, WA. (United States)

    2015-06-15

    Purpose: One of the more critical initiating events for reproductive cell death is the creation of a DNA double strand break (DSB). In this study, we present a computationally efficient way to determine spatial variations in the relative biological effectiveness (RBE) of proton therapy beams within the FLUKA Monte Carlo (MC) code. Methods: We used the independently tested Monte Carlo Damage Simulation (MCDS) developed by Stewart and colleagues (Radiat. Res. 176, 587–602 2011) to estimate the RBE for DSB induction of monoenergetic protons, tritium, deuterium, hellium-3, hellium-4 ions and delta-electrons. The dose-weighted (RBE) coefficients were incorporated into FLUKA to determine the equivalent {sup 6}°60Co γ-ray dose for representative proton beams incident on cells in an aerobic and anoxic environment. Results: We found that the proton beam RBE for DSB induction at the tip of the Bragg peak, including primary and secondary particles, is close to 1.2. Furthermore, the RBE increases laterally to the beam axis at the area of Bragg peak. At the distal edge, the RBE is in the range from 1.3–1.4 for cells irradiated under aerobic conditions and may be as large as 1.5–1.8 for cells irradiated under anoxic conditions. Across the plateau region, the recorded RBE for DSB induction is 1.02 for aerobic cells and 1.05 for cells irradiated under anoxic conditions. The contribution to total effective dose from secondary heavy ions decreases with depth and is higher at shallow depths (e.g., at the surface of the skin). Conclusion: Multiscale simulation of the RBE for DSB induction provides useful insights into spatial variations in proton RBE within pristine Bragg peaks. This methodology is potentially useful for the biological optimization of proton therapy for the treatment of cancer. The study highlights the need to incorporate spatial variations in proton RBE into proton therapy treatment plans.

  14. Becoming a crossover-competent DSB.

    Science.gov (United States)

    Lake, Cathleen M; Hawley, R Scott

    2016-06-01

    The proper execution of meiotic recombination (or crossing over) is essential for chromosome segregation during the first meiotic division, and thus this process is regulated by multiple, and often elaborate, mechanisms. Meiotic recombination begins with the programmed induction of DNA double-strand breaks (DSBs), of which only a subset are selected to be repaired into crossovers. This crossover selection process is carried out by a number of pro-crossover proteins that regulate the fashion in which DSBs are repaired. Here, we highlight recent studies regarding the process of DSB fate selection by a family of pro-crossover proteins known as the Zip-3 homologs.

  15. miR-155 Over-expression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-prone DSB Repair

    Science.gov (United States)

    Czochor, Jennifer R.; Sulkowski, Parker; Glazer, Peter M.

    2016-01-01

    miR-155 is an oncogenic microRNA (miR) that is often over-expressed in cancer and is associated with poor prognosis. miR-155 can target several DNA repair factors including RAD51, MLH1, and MSH6, and its over-expression results in an increased mutation frequency in vitro, although the mechanism has yet to be fully understood. Here, we demonstrate that over-expression of miR-155 drives an increased mutation frequency both in vitro and in vivo, promoting genomic instability by affecting multiple DNA repair pathways. miR-155 over-expression causes a decrease in homologous recombination, but yields a concurrent increase in the error-prone non-homologous end-joining (NHEJ) pathway. Despite repressing established targets MLH1 and MSH6, the identified mutation pattern upon miR-155 over-expression does not resemble that of a mismatch repair-deficient background. Further investigation revealed that all four subunits of polymerase delta, a high-fidelity DNA replication and repair polymerase, are down-regulated at the mRNA level in the context of miR-155 over-expression. FOXO3a, a transcription factor and known target of miR-155, has one or more putative binding site(s) in the promoter of all four polymerase delta subunits. Finally, suppression of FOXO3a by miR-155 or by siRNA knockdown is sufficient to repress the expression of the catalytic subunit of polymerase delta, POLD1, at the protein level, indicating that FOXO3a contributes to the regulation of polymerase delta levels. PMID:26850462

  16. DSB Tilgængelighedsstandard

    DEFF Research Database (Denmark)

    Ginnerup, Søren

    2012-01-01

    I DSB's Tilgængelighedsstandard er der lagt vægt på at tilvejebringe et overskueligt og anvendeligt værktøj for tilgængelighed til dem der skal forestå fremtidens stationsindretning i forbindelse med nybyggeri, ombygning eller modernisering af stationerne for DSB Ejendomme......I DSB's Tilgængelighedsstandard er der lagt vægt på at tilvejebringe et overskueligt og anvendeligt værktøj for tilgængelighed til dem der skal forestå fremtidens stationsindretning i forbindelse med nybyggeri, ombygning eller modernisering af stationerne for DSB Ejendomme...

  17. The disulfide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner

    OpenAIRE

    Vertommen, Didier; Depuydt, Matthieu; Pan, Jonathan; Leverrier, Pauline; Knoops, Laurent; Szikora, Jean-Pierre; Messens, Joris; Bardwell, James C. A.; Collet, Jean-Francois

    2007-01-01

    In Escherichia coli, DsbA introduces disulfide bonds into secreted proteins. DsbA is recycled by DsbB, which generates disulfides from quinone reduction. DsbA is not known to have any proofreading activity and can form incorrect disulfides in proteins with multiple cysteines. These incorrect disulfides are thought to be corrected by a protein disulfide isomerase, DsbC, which is kept in the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway is considered to be isolat...

  18. Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

    Science.gov (United States)

    Yu, Shirong; Evans, Katie; Bennett, Mark; Webster, Richard M.; Leadbitter, Matthew; Teng, Yumin; Waters, Raymond

    2016-01-01

    The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome–NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl-transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5 significantly alters the genomic distribution of NER rates; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome. PMID:27470111

  19. Molecular Understanding of Efficient DNA Repair Machinery of Photolyase

    Science.gov (United States)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2012-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA with high efficiency, through a cylic light-driven electron transfer radical mechanism. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of five active-site residues. The significant loss of repair efficiency by the mutation indicates that those active-site residues play an important role in the DNA repair by photolyase. To understand how the active-site residues modulate the efficiency, we mapped out the entire evolution of each elementary step during the repair in those photolyase mutants with femtosecond resolution. We completely analyzed the electron transfer dynamics using the Sumi-Marcus model. The results suggest that photolyase controls the critical electron transfer and the ring-splitting of pyrimidine dimer through modulation of the redox potentials and reorganization energies, and stabilization of the anionic intermediates, maintaining the dedicated balance of all the reaction steps and achieving the maximum function activity.

  20. Purification and characterization of exonuclease-free Artemis: Implications for DNA-PK – dependent processing of DNA termini in NHEJ catalyzed DSB repair

    Science.gov (United States)

    Pawelczak, Katherine S.; Turchi, John J.

    2010-01-01

    Artemis is a member of the β–CASP family of nucleases in the metallo-β-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5’ to 3’ exonuclease activity and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His]6–Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activity, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activity on a Ni-Agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. PMID:20347402

  1. Purification and characterization of exonuclease-free Artemis: Implications for DNA-PK-dependent processing of DNA termini in NHEJ-catalyzed DSB repair.

    Science.gov (United States)

    Pawelczak, Katherine S; Turchi, John J

    2010-06-04

    Artemis is a member of the beta-CASP family of nucleases in the metallo-beta-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His](6)-Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activities, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activities on a Ni-agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. Copyright 2010 Elsevier B.V. All rights reserved.

  2. Protein disulfide bond generation in Escherichia coli DsbB–DsbA

    OpenAIRE

    Inaba, Kenji

    2008-01-01

    Protein disulfide bond formation is catalyzed by a series of Dsb enzymes present in the periplasm of Escherichia coli. The crystal structure of the DsbB–DsbA–ubiquinone ternary complex provided important insights into mechanisms of the de novo disulfide bond generation cooperated by DsbB and ubiquinone and of the disulfide bond shuttle from DsbB to DsbA. The structural basis for prevention of the crosstalk between the DsbA–DsbB oxidative and the DsbC–DsbD reductive pathways has also been prop...

  3. Mutational Analysis of the Disulfide Catalysts DsbA and DsbB

    OpenAIRE

    Tan, Jacqueline; Lu, Ying; Bardwell, James C. A.

    2005-01-01

    In prokaryotes, disulfides are generated by the DsbA-DsbB system. DsbB functions to generate disulfides by quinone reduction. These disulfides are passed to the DsbA protein and then to folding proteins. To investigate the DsbA-DsbB catalytic system, we performed an in vivo selection for chromosomal dsbA and dsbB mutants. We rediscovered many residues previously shown to be important for the activity of these proteins. In addition, we obtained one novel DsbA mutant (M153R) and four novel DsbB...

  4. DsbC activation by the N-terminal domain of DsbD

    OpenAIRE

    Goldstone, David; Haebel, Peter W.; Katzen, Federico; Bader, Martin W.; Bardwell, James C. A.; Beckwith, Jon; Metcalf, Peter

    2001-01-01

    The correct formation of disulfide bonds in the periplasm of Escherichia coli involves Dsb proteins, including two related periplasmic disulfide-bond isomerases, DsbC and DsbG. DsbD is a membrane protein required to maintain the functional oxidation state of DsbC and DsbG. In this work, purified proteins were used to investigate the interaction between DsbD and DsbC. A 131-residue N-terminal fragment of DsbD (DsbDα) was expressed and purified and shown to form a fu...

  5. Expression of MLL-AF4 or AF4-MLL fusions does not impact the efficiency of DNA damage repair.

    Science.gov (United States)

    Castaño, Julio; Herrero, Ana B; Bursen, Aldeheid; González, Federico; Marschalek, Rolf; Gutiérrez, Norma C; Menendez, Pablo

    2016-05-24

    The most frequent rearrangement of the human MLL gene fuses MLL to AF4 resulting in high-risk infant B-cell acute lymphoblastic leukemia (B-ALL). MLL fusions are also hallmark oncogenic events in secondary acute myeloid leukemia. They are a direct consequence of mis-repaired DNA double strand breaks (DNA-DSBs) due to defects in the DNA damage response associated with exposure to topoisomerase-II poisons such as etoposide. It has been suggested that MLL fusions render cells susceptible to additional chromosomal damage upon exposure to etoposide. Conversely, the genome-wide mutational landscape in MLL-rearranged infant B-ALL has been reported silent. Thus, whether MLL fusions compromise the recognition and/or repair of DNA damage remains unanswered. Here, the fusion proteins MLL-AF4 (MA4) and AF4-MLL (A4M) were CRISPR/Cas9-genome edited in the AAVS1 locus of HEK293 cells as a model to study MLL fusion-mediated DNA-DSB formation/repair. Repair kinetics of etoposide- and ionizing radiation-induced DSBs was identical in WT, MA4- and A4M-expressing cells, as revealed by flow cytometry, by immunoblot for γH2AX and by comet assay. Accordingly, no differences were observed between WT, MA4- and A4M-expressing cells in the presence of master proteins involved in non-homologous end-joining (NHEJ; i.e.KU86, KU70), alternative-NHEJ (Alt-NHEJ; i.e.LigIIIa, WRN and PARP1), and homologous recombination (HR, i.e.RAD51). Moreover, functional assays revealed identical NHEJ and HR efficiency irrespective of the genotype. Treatment with etoposide consistently induced cell cycle arrest in S/G2/M independent of MA4/A4M expression, revealing a proper activation of the DNA damage checkpoints. Collectively, expression of MA4 or A4M does neither influence DNA signaling nor DNA-DSB repair.

  6. Biomarkers for DNA DSB inhibitors and radiotherapy clinical trials.

    Science.gov (United States)

    Liu, Stanley K; Olive, Peggy L; Bristow, Robert G

    2008-09-01

    Major technical advances in radiotherapy, including IMRT and image-guided radiotherapy, have allowed for improved physical precision and increased dose delivery to the tumor, with better sparing of surrounding normal tissue. The development of inhibitors of the sensing and repair of DNA double-strand breaks (DSBs) is exciting and could be combined with precise radiotherapy targeting to improve local control following radiotherapy. However, caution must be exercised in order that DSB inhibitors are combined with radiotherapy in such a manner as to preserve the therapeutic ratio by exploiting repair deficiencies in malignant cells over that of normal cells. In this review, we discuss the rationale and current approaches to targeting DSB sensing and repair pathways in combined modality with radiotherapy. We also describe potential biomarkers that could be useful in detecting functional inhibition of DSB repair in a patient's tissues during clinical radiotherapy trials. Finally, we examine a number of issues relating to the use of DSB-inhibiting molecular agents and radiotherapy in the context of the tumor microenvironment, effects on normal tissues and the optimal timing and duration of the agent in relation to fractionated radiotherapy.

  7. Transcript-RNA-templated DNA recombination and repair.

    Science.gov (United States)

    Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

    2014-11-20

    Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.

  8. The accumulation of un-repairable DNA damage in laminopathy progeria fibroblasts is caused by ROS generation and is prevented by treatment with N-acetyl cysteine.

    Science.gov (United States)

    Richards, Shane A; Muter, Joanne; Ritchie, Pamela; Lattanzi, Giovanna; Hutchison, Christopher J

    2011-10-15

    Fibroblasts from patients with the severe laminopathy diseases, restrictive dermopathy (RD) and Hutchinson Gilford progeria syndrome (HGPS), are characterized by poor growth in culture, the presence of abnormally shaped nuclei and the accumulation of DNA double-strand breaks (DSB). Here we show that the accumulation of DSB and poor growth of the fibroblasts but not the presence of abnormally shaped nuclei are caused by elevated levels of reactive oxygen species (ROS) and greater sensitivity to oxidative stress. Basal levels of ROS and sensitivity to H(2)O(2) were compared in fibroblasts from normal, RD and HGPS individuals using fluorescence activated cell sorting-based assays. Basal levels of ROS and stimulated levels of ROS were both 5-fold higher in the progeria fibroblasts. Elevated levels of ROS were correlated with lower proliferation indices but not with the presence of abnormally shaped nuclei. DSB induced by etoposide were repaired efficiently in normal, RD and HGPS fibroblasts. In contrast, DSB induced by ROS were repaired efficiently in normal fibroblasts, but in RD and HGPS fibroblasts many ROS-induced DSB were un-repairable. The accumulation of ROS-induced DSB appeared to cause the poor growth of RD and HGPS fibroblasts, since culture in the presence of the ROS scavenger N-acetyl cysteine (NAC) reduced the basal levels of DSB, eliminated un-repairable ROS-induced DSB and greatly improved population-doubling times. Our findings suggest that un-repaired ROS-induced DSB contribute significantly to the RD and HGPS phenotypes and that inclusion of NAC in a combinatorial therapy might prove beneficial to HGPS patients.

  9. Mouse RAD54 Affects DNA Double-Strand Break Repair and Sister Chromatid Exchange

    Science.gov (United States)

    Dronkert, Mies L. G.; Beverloo, H. Berna; Johnson, Roger D.; Hoeijmakers, Jan H. J.; Jasin, Maria; Kanaar, Roland

    2000-01-01

    Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA. PMID:10757799

  10. DNA-DSB in CHO-K1 cells induced by heavy-ions: Break rejoining and residual damage (GSI)

    Science.gov (United States)

    Taucher-Scholz, G.; Heilmann, J.; Becher, G.; Kraft, G.

    1994-01-01

    DNA double strand breaks (DSB's) are the critical lesions involved in cellular effects of ionizing radiation. Therefore, the evaluation of DSB induction in mammalian cells after heavy ion irradiation is an essential task for the assessment of high-LET radiation risk in space. Of particular interest has been the question of how the biological efficiency for the cellular inactivation endpoint relates to the initial lesions (DSBs) at varying LETs. For cell killing, an increased Relative Biological Efficiency (RBE) has been determined for highLET radiation around 100-200 keV/mu m. At higher LET, the RBE's decrease again to values below one for the very heavy particles. At GSI, DSB-induction was measured in CHO-K1 cells following irradiation with accelerated particles covering a wide LET range. The electrophoretic elution of fragmented DNA out of agarose plugs in a constant electrical field was applied for the detection of DSB's. The fraction of DNA retained was determined considering the relative intensities of ethidium bromide fluorescence in the well and in the gel lane. Dose-effect curves were established, from which the RBE for DSB induction was calculated at a fraction of 0.7 of DNA retained In summary, these rejoining studies are in line with an enhanced severity of the DNA DSB's at higher LET's, resulting in a decreased repairability of the induced lesions. However, no information concerning the fidelity of strand breaks rejoining is provided in these studies. To assess correct rejoining of DNA fragments an experimental system involving individual DNA hybridization bands has been set up. In preliminary experiments Sal I generated DNA fragments of 0.9 Mbp were irradiated with xrays and incubated for repair However, restitution of the original signals was not observed, probably due to the high radiation dose necessary for breakage of a fragment of this size. A banding pattern with NotI hybridization signals in a higher MW range (3Mbp) has been obtained by varying

  11. Transcript RNA supports precise repair of its own DNA gene.

    Science.gov (United States)

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

    The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

  12. Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Farjana Fattah

    2010-02-01

    Full Text Available The repair of DNA double-strand breaks (DSBs is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR and non-homologous end joining (NHEJ. In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways-the main Ku heterodimer-dependent or "classic" NHEJ (C-NHEJ pathway and an "alternative" NHEJ (A-NHEJ pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PK(cs, XLF, and LIGIV, and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PK(cs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PK(cs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

  13. Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Farjana Fattah

    2010-02-01

    Full Text Available The repair of DNA double-strand breaks (DSBs is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR and non-homologous end joining (NHEJ. In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways-the main Ku heterodimer-dependent or "classic" NHEJ (C-NHEJ pathway and an "alternative" NHEJ (A-NHEJ pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PK(cs, XLF, and LIGIV, and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PK(cs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PK(cs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

  14. Non-DSB clustered DNA lesions. Does theory colocalize with the experiment?

    Science.gov (United States)

    Nikitaki, Zacharenia; Nikolov, Vladimir; Mavragani, Ifigeneia V.; Plante, Ianik; Emfietzoglou, Dimitris; Iliakis, George; Georgakilas, Alexandros G.

    2016-11-01

    Ionizing radiation results in various kinds of DNA lesions such as double strand breaks (DSBs) and other non-DSB base lesions. These lesions may be formed in close proximity (i.e., within a few nanometers) resulting in clustered types of DNA lesions. These damage clusters are considered the fingerprint of ionizing radiation, notably charged particles of high linear energy transfer (LET). Accumulating theoretical and experimental evidence suggests that the induction of these clustered lesions appears under various irradiation conditions but also as a result of high levels of oxidative stress. The biological significance of these clustered DNA lesions pertains to the inability of cells to process them efficiently compared to isolated DNA lesions. The results in the case of unsuccessful or erroneous repair can vary from mutations up to chromosomal instability. In this mini review, we discuss of several Monte Carlo simulations codes and experimental evidence regarding the induction and repair of radiation-induced non-DSB complex DNA lesions. We also critically present the most widely used methodologies (i.e., gel electrophoresis and fluorescence microscopy [in situ colocalization assays]). Based on the comparison of different approaches, we provide examples and suggestions for the improved detection of these lesions in situ. Based on the current status of knowledge, we conclude that there is a great need for improvement of the detection techniques at the cellular or tissue level, which will provide valuable information for understanding the mechanisms used by the cell to process clustered DNA lesions.

  15. Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

  16. PHF11 promotes DSB resection, ATR signaling, and HR

    Science.gov (United States)

    Gong, Yi; Handa, Naofumi; Kowalczykowski, Stephen C.; de Lange, Titia

    2017-01-01

    Resection of double-strand breaks (DSBs) plays a critical role in their detection and appropriate repair. The 3′ ssDNA protrusion formed through resection activates the ATR-dependent DNA damage response (DDR) and is required for DSB repair by homologous recombination (HR). Here we report that PHF11 (plant homeodomain finger 11) encodes a previously unknown DDR factor involved in 5′ end resection, ATR signaling, and HR. PHF11 was identified based on its association with deprotected telomeres and localized to sites of DNA damage in S phase. Depletion of PHF11 diminished the ATR signaling response to telomere dysfunction and genome-wide DNA damage, reduced end resection at sites of DNA damage, resulted in compromised HR and misrejoining of S-phase DSBs, and increased the sensitivity to DNA-damaging agents. PHF11 interacted with the ssDNA-binding protein RPA and was found in a complex with several nucleases, including the 5′ dsDNA exonuclease EXO1. Biochemical experiments demonstrated that PHF11 stimulates EXO1 by overcoming its inhibition by RPA, suggesting that PHF11 acts (in part) by promoting 5′ end resection at RPA-bound sites of DNA damage. These findings reveal a role for PHF11 in DSB resection, DNA damage signaling, and DSB repair. PMID:28115467

  17. PHF11 promotes DSB resection, ATR signaling, and HR.

    Science.gov (United States)

    Gong, Yi; Handa, Naofumi; Kowalczykowski, Stephen C; de Lange, Titia

    2017-01-01

    Resection of double-strand breaks (DSBs) plays a critical role in their detection and appropriate repair. The 3' ssDNA protrusion formed through resection activates the ATR-dependent DNA damage response (DDR) and is required for DSB repair by homologous recombination (HR). Here we report that PHF11 (plant homeodomain finger 11) encodes a previously unknown DDR factor involved in 5' end resection, ATR signaling, and HR. PHF11 was identified based on its association with deprotected telomeres and localized to sites of DNA damage in S phase. Depletion of PHF11 diminished the ATR signaling response to telomere dysfunction and genome-wide DNA damage, reduced end resection at sites of DNA damage, resulted in compromised HR and misrejoining of S-phase DSBs, and increased the sensitivity to DNA-damaging agents. PHF11 interacted with the ssDNA-binding protein RPA and was found in a complex with several nucleases, including the 5' dsDNA exonuclease EXO1. Biochemical experiments demonstrated that PHF11 stimulates EXO1 by overcoming its inhibition by RPA, suggesting that PHF11 acts (in part) by promoting 5' end resection at RPA-bound sites of DNA damage. These findings reveal a role for PHF11 in DSB resection, DNA damage signaling, and DSB repair.

  18. Changes in DNA repair during aging

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei; Mao, Zhiyong; Hine, Christpher

    2007-01-01

    DNA is a precious molecule. It encodes vital information about cellular content and function. There are only two copies of each chromosome in the cell, and once the sequence is lost no replacement is possible. The irreplaceable nature of the DNA sets it apart from other cellular molecules, and makes it a critical target for age-related deterioration. To prevent DNA damage cells have evolved elaborate DNA repair machinery. Paradoxically, DNA repair can itself be subject to age-related changes and deterioration. In this review we will discuss the changes in efficiency of mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER) and double-strand break (DSB) repair systems during aging, and potential changes in DSB repair pathway usage that occur with age. Mutations in DNA repair genes and premature aging phenotypes they cause have been reviewed extensively elsewhere, therefore the focus of this review is on the comparison of DNA repair mechanisms in young versus old. PMID:17913742

  19. DNA double-strand break repair is involved in desiccation resistance of Sinorhizobium meliloti, but is not essential for its symbiotic interaction with Medicago truncatula.

    Science.gov (United States)

    Dupuy, Pierre; Gourion, Benjamin; Sauviac, Laurent; Bruand, Claude

    2016-11-23

    The soil bacterium Sinorhizobium meliloti, a nitrogen-fixing symbiont of legume plants, is exposed to numerous stress conditions in nature, some of which cause the formation of harmful DNA double strand breaks (DSB). In particular, the reactive oxygen (ROS) and nitrogen (RNS) species produced during symbiosis, and the desiccation occurring in dry soils, are conditions which induce DSB. Two major systems of DSB repair are known in S. meliloti: homologous recombination (HR) and non-homologous end-joining (NHEJ). However, their role in the resistance to ROS, RNS and desiccation has never been examined in this bacterial species, and the importance of DSB repair in the symbiotic interaction has not been properly evaluated. Here, we constructed S. meliloti strains deficient in HR (by deleting the recA gene) or in NHEJ (by deleting the four ku genes) or both. Interestingly, we observed that ku and/or recA genes are involved in S. meliloti resistance to ROS and RNS. Nevertheless, a S. meliloti strain deficient in both HR and NHEJ was not altered in its ability to establish and maintain an efficient nitrogen-fixing symbiosis with Medicago truncatula, showing that rhizobial DSB repair is not essential for this process. This result suggests either that DSB formation in S. meliloti is efficiently prevented during symbiosis, or that DSB are not detrimental for symbiosis efficiency. In contrast, we found for the first time that both recA and ku genes are involved in S. meliloti resistance to desiccation, suggesting that DSB repair could be important for rhizobium persistence in the soil.

  20. Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

    Science.gov (United States)

    Cucinotta, F. A.; Nikjoo, H.; O'Neill, P.; Goodhead, D. T.

    2000-01-01

    PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

  1. Efficient and reproducible identification of mismatch repair deficient colon cancer

    DEFF Research Database (Denmark)

    Joost, Patrick; Bendahl, Pär-Ola; Halvarsson, Britta;

    2013-01-01

    BACKGROUND: The identification of mismatch-repair (MMR) defective colon cancer is clinically relevant for diagnostic, prognostic and potentially also for treatment predictive purposes. Preselection of tumors for MMR analysis can be obtained with predictive models, which need to demonstrate ease...... of application and favorable reproducibility. METHODS: We validated the MMR index for the identification of prognostically favorable MMR deficient colon cancers and compared performance to 5 other prediction models. In total, 474 colon cancers diagnosed ≥ age 50 were evaluated with correlation between...... and efficiently identifies MMR defective colon cancers with high sensitivity and specificity. The model shows stable performance with low inter-observer variability and favorable performance when compared to other MMR predictive models....

  2. Energy Efficiency and Air Quality Repairs at Lyonsdale Biomass

    Energy Technology Data Exchange (ETDEWEB)

    Brower, Michael R; Morrison, James A; Spomer, Eric; Thimot, Carol A

    2012-07-31

    This project enabled Lyonsdale Biomass, LLC to effect analyses, repairs and upgrades for its biomass cogeneration facility located in Lewis County, New York and close by the Adirondack Park to reduce air emissions by improving combustion technique and through the overall reduction of biomass throughput by increasing the system's thermodynamic efficiency for its steam-electrical generating cycle. Project outcomes result in significant local, New York State, Northeast U.S. and national benefits including improved renewable energy operational surety, enhanced renewable energy efficiency and more freedom from foreign fossil fuel source dependence. Specifically, the reliability of the Lyonsdale Biomass 20MWe woody biomass combined-heat and power (CHP) was and is now directly enhanced. The New York State and Lewis County benefits are equally substantial since the facility sustains 26 full-time equivalency (FTE) jobs at the facility and as many as 125 FTE jobs in the biomass logistics supply chain. Additionally, the project sustains essential local and state payment in lieu of taxes revenues. This project helps meet several USDOE milestones and contributes directly to the following sustainability goals:  Climate: Reduces greenhouse gas emissions associated with bio-power production, conversion and use, in comparison to fossil fuels. Efficiency and Productivity: Enhances efficient use of renewable resources and maximizes conversion efficiency and productivity. Profitability: Lowers production costs. Rural Development: Enhances economic welfare and rural development through job creation and income generation. Standards: Develop standards and corresponding metrics for ensuring sustainable biopower production. Energy Diversification and Security: Reduces dependence on foreign oil and increases energy supply diversity. Net Energy Balance: Ensures positive net energy balance for all alternatives to fossil fuels.

  3. Alternative end-joining and classical nonhomologous end-joining pathways repair different types of double-strand breaks during class-switch recombination.

    Science.gov (United States)

    Cortizas, Elena M; Zahn, Astrid; Hajjar, Maurice E; Patenaude, Anne-Marie; Di Noia, Javier M; Verdun, Ramiro E

    2013-12-01

    Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ-defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.

  4. Homeostatic regulation of meiotic DSB formation by ATM/ATR

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Tim J.; Wardell, Kayleigh; Garcia, Valerie; Neale, Matthew J., E-mail: m.neale@sussex.ac.uk

    2014-11-15

    Ataxia–telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.

  5. Homeostatic regulation of meiotic DSB formation by ATM/ATR.

    Science.gov (United States)

    Cooper, Tim J; Wardell, Kayleigh; Garcia, Valerie; Neale, Matthew J

    2014-11-15

    Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.

  6. Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.

    Directory of Open Access Journals (Sweden)

    Tamara Goldfarb

    Full Text Available Recombination between homologous chromosomes of different parental origin (homologs is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs] show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in

  7. Overexpression of OsRecQl4 and/or OsExo1 enhances DSB-induced homologous recombination in rice.

    Science.gov (United States)

    Kwon, Yong-Ik; Abe, Kiyomi; Osakabe, Keishi; Endo, Masaki; Nishizawa-Yokoi, Ayako; Saika, Hiroaki; Shimada, Hiroaki; Toki, Seiichi

    2012-12-01

    During homologous recombination (HR)-mediated DNA double-strand break (DSB) repair in eukaryotes, an initial step is the creation of a 3'-single-stranded DNA (ssDNA) overhang via resection of a 5' end. Rad51 polymerizes on this ssDNA to search for a homologous sequence, and the gapped sequence is then repaired using an undamaged homologous DNA strand as template. Recent studies in eukaryotes indicate that resection of the DSB site is promoted by the cooperative action of RecQ helicase family proteins: Bloom helicase (BLM) in mammals or Sgs1 in yeast, and exonuclease 1 (Exo1). However, the role of RecQ helicase and exonuclease during the 5'-resection process of HR in plant cells has not yet been defined. Here, we demonstrate that overexpression of rice proteins OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance DSB processing, as evaluated by recombination substrate reporter lines in rice. These results could be applied to construct an efficient gene targeting system in rice.

  8. Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB induction and conversion induced by gamma rays and helium ions.

    Science.gov (United States)

    Tsai, Ju-Ying; Chen, Fang-Hsin; Hsieh, Tsung-Yu; Hsiao, Ya-Yun

    2015-07-01

    Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/μm) to (60)Co gamma rays were 3.0 and 3.2, respectively. These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was ∼1-4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing.

  9. Gene Targeting Without DSB Induction Is Inefficient in Barley.

    Science.gov (United States)

    Horvath, Mihaly; Steinbiss, Hans-Henning; Reiss, Bernd

    2016-01-01

    Double strand-break (DSB) induction allowed efficient gene targeting in barley (Hordeum vulgare), but little is known about efficiencies in its absence. To obtain such data, an assay system based on the acetolactate synthase (ALS) gene was established, a target gene which had been used previously in rice and Arabidopsis thaliana. Expression of recombinases RAD51 and RAD54 had been shown to improve gene targeting in A. thaliana and positive-negative (P-N) selection allows the routine production of targeted mutants without DSB induction in rice. We implemented these approaches in barley and analysed gene targeting with the ALS gene in wild type and RAD51 and RAD54 transgenic lines. In addition, P-N selection was tested. In contrast to the high gene targeting efficiencies obtained in the absence of DSB induction in A. thaliana or rice, not one single gene targeting event was obtained in barley. These data suggest that gene targeting efficiencies are very low in barley and can substantially differ between different plants, even at the same target locus. They also suggest that the amount of labour and time would become unreasonably high to use these methods as a tool in routine applications. This is particularly true since DSB induction offers efficient alternatives. Barley, unlike rice and A. thaliana has a large, complex genome, suggesting that genome size or complexity could be the reason for the low efficiencies. We discuss to what extent transformation methods, genome size or genome complexity could contribute to the striking differences in the gene targeting efficiencies between barley, rice and A. thaliana.

  10. Structure-specific endonucleases Xpf and Mus81 play overlapping but essential roles in DNA repair by homologous recombination

    NARCIS (Netherlands)

    K. Kikuchi (Koji); T. Narita (Takeo); V.C. Pham (Van Ca); J. Iijima (Junko); T. Hirota (Tomomitsu); I.S. Keka (Islam Shamima); Mohiuddin; K. Okawa (Katsuya); T. Hori (Toshiyuki); T. Fukagawa (Tatsuo); J. Essers (Jeroen); R. Kanaar (Roland); M.C. Whitby (Matthew); K. Sugasawa (Kaoru); Y. Taniguchi (Yoshihito); K. Kitagawa; S. Takeda (Shiunichi)

    2013-01-01

    textabstractDNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intac

  11. Structure-specific endonucleases Xpf and Mus81 play overlapping but essential roles in DNA repair by homologous recombination

    NARCIS (Netherlands)

    K. Kikuchi (Koji); T. Narita (Takeo); V.C. Pham (Van Ca); J. Iijima (Junko); T. Hirota (Tomomitsu); I.S. Keka (Islam Shamima); Mohiuddin; K. Okawa (Katsuya); T. Hori (Toshiyuki); T. Fukagawa (Tatsuo); J. Essers (Jeroen); R. Kanaar (Roland); M.C. Whitby (Matthew); K. Sugasawa (Kaoru); Y. Taniguchi (Yoshihito); K. Kitagawa; S. Takeda (Shiunichi)

    2013-01-01

    textabstractDNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intac

  12. Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

    Science.gov (United States)

    Kouass Sahbani, S; Rezaee, M; Cloutier, P; Sanche, L; Hunting, D J

    2014-06-25

    The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

  13. Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair

    NARCIS (Netherlands)

    N. Hermans (Nicolaas); C. Laffeber; M. Cristovao (Michele); Artola-Borán, M. (Mariela); Mardenborough, Y. (Yannicka); P. Ikpa (Pauline); Jaddoe, A. (Aruna); H.H.K. Winterwerp (Herrie); C. Wyman (Claire); J. Jiricny (Josef); R. Kanaar (Roland); P. Friedhoff (Peter); J.H.G. Lebbink (Joyce)

    2016-01-01

    textabstractDNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of th

  14. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels.

    Science.gov (United States)

    Joshi, Neeraj; Brown, M Scott; Bishop, Douglas K; Börner, G Valentin

    2015-03-05

    During meiosis, Spo11-induced double-strand breaks (DSBs) are processed into crossovers, ensuring segregation of homologous chromosomes (homologs). Meiotic DSB processing entails 5' end resection and preferred strand exchange with the homolog rather than the sister chromatid (homolog bias). In many organisms, DSBs appear gradually along the genome. Here we report unexpected effects of global DSB levels on local recombination events. Early-occurring, low-abundance "scout" DSBs lack homolog bias. Their resection and interhomolog processing are controlled by the conserved checkpoint proteins Tel1(ATM) kinase and Pch2(TRIP13) ATPase. Processing pathways controlled by Mec1(ATR) kinase take over these functions only above a distinct DSB threshold, resulting in progressive strengthening of the homolog bias. We conclude that Tel1(ATM)/Pch2 and Mec1(ATR) DNA damage response pathways are sequentially activated during wild-type meiosis because of their distinct sensitivities to global DSB levels. Moreover, relative DSB order controls the DSB repair pathway choice and, ultimately, recombination outcome.

  15. DsbL and DsbI contribute to periplasmic disulfide bond formation in Salmonella enterica serovar Typhimurium

    OpenAIRE

    Lin, Dongxia; Kim, Byoungkwan; Slauch, James M.

    2009-01-01

    Disulfide bond formation in periplasmic proteins is catalysed by the DsbA/DsbB system in most Gram-negative bacteria. Salmonella enterica serovar Typhimurium also encodes a paralogous pair of proteins to DsbA and DsbB, DsbL and DsbI, respectively, downstream of a periplasmic arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI function as a redox pair contributing to periplasmic disulfide bond formation and, as such, affect transcription of the Salmonella pathogenicity island 1 (SP...

  16. Rad52 SUMOylation affects the efficiency of the DNA repair

    DEFF Research Database (Denmark)

    Altmannova, Veronika; Eckert-Boulet, Nadine; Arneric, Milica

    2010-01-01

    Homologous recombination (HR) plays a vital role in DNA metabolic processes including meiosis, DNA repair, DNA replication and rDNA homeostasis. HR defects can lead to pathological outcomes, including genetic diseases and cancer. Recent studies suggest that the post-translational modification...... recombination mediator protein Rad52. Interestingly, Rad52 SUMOylation is enhanced by single-stranded DNA, and we show that SUMOylation of Rad52 also inhibits its DNA binding and annealing activities. The biochemical effects of SUMO modification in vitro are accompanied by a shorter duration of spontaneous Rad...... of recombination and DNA repair....

  17. OsSDS is essential for DSB formation in rice meiosis

    Directory of Open Access Journals (Sweden)

    Zhigang eWu

    2015-02-01

    Full Text Available SDS is a meiosis specific cyclin-like protein and required for DMC1 mediated double-strand break (DSB repairing in Arabidopsis. Here, we found its rice homolog, OsSDS, is essential for meiotic DSB formation. The Ossds mutant is normal in vegetative growth but both male and female gametes are inviable. The Ossds meiocytes exhibit severe defects in homologous pairing and synapsis. No γH2AX immunosignals in Ossds meiocytes together with the suppression of chromosome fragmentation in Ossds-1 Osrad51c, both provide strong evidences that OsSDS is essential for meiotic DSB formation. Immunostaining investigations revealed that meiotic chromosome axes are normally formed but both SC installation and localization of recombination elements are failed in Ossds. We suspected that this cyclin protein has been differentiated pretty much between monocots and dicots on its function in meiosis.

  18. OsSDS is essential for DSB formation in rice meiosis.

    Science.gov (United States)

    Wu, Zhigang; Ji, Jianhui; Tang, Ding; Wang, Hongjun; Shen, Yi; Shi, Wenqing; Li, Yafei; Tan, Xuelin; Cheng, Zhukuan; Luo, Qiong

    2015-01-01

    SDS is a meiosis specific cyclin-like protein and required for DMC1 mediated double-strand break (DSB) repairing in Arabidopsis. Here, we found its rice homolog, OsSDS, is essential for meiotic DSB formation. The Ossds mutant is normal in vegetative growth but both male and female gametes are inviable. The Ossds meiocytes exhibit severe defects in homologous pairing and synapsis. No γH2AX immunosignals in Ossds meiocytes together with the suppression of chromosome fragmentation in Ossds-1 Osrad51c, both provide strong evidences that OsSDS is essential for meiotic DSB formation. Immunostaining investigations revealed that meiotic chromosome axes are normally formed but both SC installation and localization of recombination elements are failed in Ossds. We suspected that this cyclin protein has been differentiated pretty much between monocots and dicots on its function in meiosis.

  19. Heavy ion induced DNA-DSB in yeast and mammalian cells

    Science.gov (United States)

    Loebrich, M.; Ikpeme, S.; Kiefer, J.

    1994-01-01

    Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.

  20. The spatial regulation of meiotic recombination hotspots: are all DSB hotspots crossover hotspots?

    Science.gov (United States)

    Serrentino, Maria-Elisabetta; Borde, Valérie

    2012-07-15

    A key step for the success of meiosis is programmed homologous recombination, during which crossovers, or exchange of chromosome arms, take place. Crossovers increase genetic diversity but their main function is to ensure accurate chromosome segregation. Defects in crossover number and position produce aneuploidies that represent the main cause of miscarriages and chromosomal abnormalities such as Down's syndrome. Recombination is initiated by the formation of programmed double strand breaks (DSBs), which occur preferentially at places called DSB hotspots. Among all DSBs generated, only a small fraction is repaired by crossover, the other being repaired by other homologous recombination pathways. Crossover maps have been generated in a number of organisms, defining crossover hotspots. With the availability of genome-wide maps of DSBs as well as the ability to measure genetically the repair outcome at several hotspots, it is becoming more and more clear that not all DSB hotspots behave the same for crossover formation, suggesting that chromosomal features distinguish different types of hotspots.

  1. STUDY OF THE PARAMETERS OF EFFICIENCY IN CENTRES FOR REPAIR OF AGRICULTURAL MACHINERY

    Directory of Open Access Journals (Sweden)

    Natalia Stoyanova

    2015-06-01

    Full Text Available The paper makes a thorough study of the parameters of efficiency in the centers for the repair of agricultural machinery, considering production and technological structure, the basic principles for design of the process of service, the quantitative indicators for servicing. It presents a theoretical model for the management of services in the service business, taking into account the basic system requirements for maintenance of agricultural machinery, the main elements of the standards of customer service, choice of forms for maintenance of agricultural machinery. Opportunities are proposed for the optimization of repair activities in the centers for repair of agricultural machinery.

  2. RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends.

    Directory of Open Access Journals (Sweden)

    Jim Westmoreland

    2009-09-01

    Full Text Available Resection of DNA double-strand break (DSB ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random "dirty-ended" DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE. We utilized this "PFGE-shift" to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after gamma-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1-2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.

  3. RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends.

    Directory of Open Access Journals (Sweden)

    Jim Westmoreland

    2009-09-01

    Full Text Available Resection of DNA double-strand break (DSB ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random "dirty-ended" DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE. We utilized this "PFGE-shift" to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after gamma-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1-2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.

  4. RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

    Science.gov (United States)

    Westmoreland, Jim; Ma, Wenjian; Yan, Yan; Van Hulle, Kelly; Malkova, Anna; Resnick, Michael A.

    2009-01-01

    Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent. PMID:19763170

  5. A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae.

    Science.gov (United States)

    McKinney, Jennifer Summers; Sethi, Sunaina; Tripp, Jennifer DeMars; Nguyen, Thuy N; Sanderson, Brian A; Westmoreland, James W; Resnick, Michael A; Lewis, L Kevin

    2013-04-15

    Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown. To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions. We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.

  6. New tools to study DNA double-strand break repair pathway choice.

    Directory of Open Access Journals (Sweden)

    Daniel Gomez-Cabello

    Full Text Available A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences.

  7. ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

    OpenAIRE

    Kijas, Amanda W.; Lim, Yi Chieh; Bolderson, Emma; Cerosaletti, Karen; Gatei, Magtouf; Jakob, Burkhard; Tobias, Frank; Taucher-Scholz, Gisela; Gueven, Nuri; Oakley, Greg; Concannon, Patrick; Wolvetang, Ernst; Khanna, Kum Kum; Wiesmüller, Lisa; Lavin, Martin F.

    2015-01-01

    The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the ...

  8. HTP-3 links DSB formation with homolog pairing and crossing over during C. elegans meiosis.

    Science.gov (United States)

    Goodyer, William; Kaitna, Susanne; Couteau, Florence; Ward, Jordan D; Boulton, Simon J; Zetka, Monique

    2008-02-01

    Repair of the programmed meiotic double-strand breaks (DSBs) that initiate recombination must be coordinated with homolog pairing to generate crossovers capable of directing chromosome segregation. Chromosome pairing and synapsis proceed independently of recombination in worms and flies, suggesting a paradoxical lack of coregulation. Here, we find that the meiotic axis component HTP-3 links DSB formation with homolog pairing and synapsis. HTP-3 forms complexes with the DSB repair components MRE-11/RAD-50 and the meiosis-specific axis component HIM-3. Loss of htp-3 or mre-11 recapitulates meiotic phenotypes consistent with a failure to generate DSBs, suggesting that HTP-3 associates with MRE-11/RAD-50 in a complex required for meiotic DSB formation. Loss of HTP-3 eliminates HIM-3 localization to axes and HIM-3-dependent homolog alignment, synapsis, and crossing over. Our study reveals a mechanism for coupling meiotic DSB formation with homolog pairing through the essential participation of an axis component with complexes mediating both processes.

  9. Conservative Repair of a Chromosomal Double-Strand Break by Single-Strand DNA through Two Steps of Annealing▿ †

    OpenAIRE

    Storici, Francesca; Snipe, Joyce R.; Chan, Godwin K.; Dmitry A Gordenin; Michael A Resnick

    2006-01-01

    The repair of chromosomal double-strand breaks (DSBs) is essential to normal cell growth, and homologous recombination is a universal process for DSB repair. We explored DSB repair mechanisms in the yeast Saccharomyces cerevisiae using single-strand oligonucleotides with homology to both sides of a DSB. Oligonucleotide-directed repair occurred exclusively via Rad52- and Rad59-mediated single-strand annealing (SSA). Even the SSA domain of human Rad52 provided partial complementation for a null...

  10. Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Mezard, C.; Nicolas, A. [Universite Paris-Sud, Orsay (France)

    1994-02-01

    Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions; (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product. 67 refs., 4 figs., 5 tabs.

  11. Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants.

    Directory of Open Access Journals (Sweden)

    Bret R Adams

    Full Text Available The DNA double-strand break (DSB is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ is dominant. We have characterized the DNA damage response (DDR and quality of DNA double-strand break (DSB repair in human embryonic stem cells (hESCs, and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR, showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ] in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

  12. Meiotic and mitotic functions of mammalian RAD 18 in DNA double-strand break repair

    NARCIS (Netherlands)

    A. Inagaki (Akiko)

    2010-01-01

    textabstractThis thesis focuses on the role of RAD 18 in DNA double-strand break (DSB ) repair. Much is known about the role of RAD 18, and its critical substrate PCNA in replication damage bypass (RDB ) repair. However, the roles of RAD 18 in DSB repair are still elusive, although several

  13. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  14. Meiotic and mitotic functions of mammalian RAD 18 in DNA double-strand break repair

    NARCIS (Netherlands)

    A. Inagaki (Akiko)

    2010-01-01

    textabstractThis thesis focuses on the role of RAD 18 in DNA double-strand break (DSB ) repair. Much is known about the role of RAD 18, and its critical substrate PCNA in replication damage bypass (RDB ) repair. However, the roles of RAD 18 in DSB repair are still elusive, although several interacti

  15. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  16. Beyond repair foci: DNA double-strand break repair in euchromatic and heterochromatic compartments analyzed by transmission electron microscopy.

    Directory of Open Access Journals (Sweden)

    Yvonne Lorat

    Full Text Available PURPOSE: DNA double-strand breaks (DSBs generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair. METHODS AND MATERIALS: Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent and heterochromatin (electron-dense in cortical neurons of irradiated mouse brain. RESULTS: While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥ 6 gold beads may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥ 10 gold beads, occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage. DISCUSSION: Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more

  17. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

    DEFF Research Database (Denmark)

    Gao, Min; Wei, Wei; Li, Ming Hua

    2014-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute...... (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian...

  18. Expression on three dsb genes of Chlamydia trachomatis serovar F

    Institute of Scientific and Technical Information of China (English)

    XING DONG YE; LI SHEN; YOU XUN ZHANG

    2007-01-01

    To investigate the expression levels of three Dsb protein genes, dsbB, dsbD and dsbG, at different time points post C. trachomatis infection, mouse fibroblast L2 cells were chosen to be infected with C. trachomatis serovar F strain F/IC-Cal-13. C. trachomatis elementary body (EB)-infected L2cells were harvested immediately after EB attachment onto the cells and every 4 hours post infection (hpi)till 44 hpi for total RNA preparation. RT-PCR assays were then employed to amplify cDNA with primer pairs which are specific to C. trachomatis dsb genes dsbB, dsbD, dsbG and tufA respectively. The relative expression levels of Dsb protein genes were evaluated as cDNA ratios of gene dsb to gene tufA. Our results showed that the transcription of dsbG started from 12 hpi and gradually increased till 44 hpi. The transcription of dsbB and dsbD were detected at 16 hpi and reached their peaks at 28 hpi and 24-28 hpi,respectively. Moreover, there was obvious transcription of dsbB at the later stage (44 hpi), but none for dsbD at this time point. We came to the conclusion that the expression levels of the three Dsb protein genes are different during the developmental cycle of C. trachomatist. They may play a role in mid-to-late stage of the developmental cycle of C. trachomatis.

  19. Dynamics and mechanism of UV-damaged DNA repair in indole-thymine dimer adduct: molecular origin of low repair quantum efficiency.

    Science.gov (United States)

    Guo, Xunmin; Liu, Zheyun; Song, Qinhua; Wang, Lijuan; Zhong, Dongping

    2015-02-26

    Many biomimetic chemical systems for repair of UV-damaged DNA showed very low repair efficiency, and the molecular origin is still unknown. Here, we report our systematic characterization of the repair dynamics of a model compound of indole-thymine dimer adduct in three solvents with different polarity. By resolving all elementary steps including three electron-transfer processes and two bond-breaking and bond-formation dynamics with femtosecond resolution, we observed the slow electron injection in 580 ps in water, 4 ns in acetonitrile, and 1.38 ns in dioxane, the fast back electron transfer without repair in 120, 150, and 180 ps, and the slow bond splitting in 550 ps, 1.9 ns, and 4.5 ns, respectively. The dimer bond cleavage is clearly accelerated by the solvent polarity. By comparing with the biological repair machine photolyase with a slow back electron transfer (2.4 ns) and a fast bond cleavage (90 ps), the low repair efficiency in the biomimetic system is mainly determined by the fast back electron transfer and slow bond breakage. We also found that the model system exists in a dynamic heterogeneous C-clamped conformation, leading to a stretched dynamic behavior. In water, we even identified another stacked form with ultrafast cyclic electron transfer, significantly reducing the repair efficiency. Thus, the comparison of the repair efficiency in different solvents is complicated and should be cautious, and only the dynamics by resolving all elementary steps can finally determine the total repair efficiency. Finally, we use the Marcus electron-transfer theory to analyze all electron-transfer reactions and rationalize all observed electron-transfer dynamics.

  20. Xbp1-mediated histone H4 deacetylation contributes to DNA double-strand break repair in yeast

    Institute of Scientific and Technical Information of China (English)

    Ran Tao; Hua Chen; Chan Gao; Pcng Xue; Fuquan Yang; Jing-Dong J Han; Bing Zhou; Ye-Guang Chen

    2011-01-01

    Xbp1 has been shown to regulate the cell cycle as a transcriptional repressor in budding yeast Saccharomyces cerevisiae.In this study,we demonstrated that Xbp1 regulates DNA double-strand break (DSB) repair in S.cerevisiae.Xbp1 physically and genetically interacts with the histone deacetylase Rpd3 complex.Chromatin immunoprecipitation revealed that Xbp1 is required for efficient deacetylation of histone H4 flanking DSBs by the Rpd3 complex.Deletion of XBP1 leads to the delayed deacetylation of histone H4,which is coupled with increased nucleosome displacement,increased DNA end resection and decreased non-homologous end-joining (NHEJ).In response to DNA damage,Xbp1 is upregulated in a Mec1-Rad9-Rad53 checkpoint pathway-dependent manner and undergoes dephosphorylation.Cdk1,a central regulator of S.cerevisiae cell cycle,is responsible for Xbp1 phosphorylation at residues Ser146,Ser271 and Ser551.Substitution of these serine residues with alanine not only increases the association of Xbp1 with the Rpd3 complex and its recruitment to a DSB,but also promotes DSB repair.Together,our findings reveal a role for Xbp1 in DSB repair via NHEJ through regulation of histone H4 acetylation and nucleosome displacement in a positive feedback manner.

  1. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells

    Science.gov (United States)

    Mao, Zhiyong; Bozzella, Michael; Seluanov, Andrei; Gorbunova, Vera

    2009-01-01

    DNA double-strand breaks (DSBs) are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSBs are nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is an intrinsically error-prone pathway while HR results in accurate repair. To understand the origin of genomic instability in human cells it is important to know the contribution of each DSB repair pathway. Studies of rodent cells and human cancer cell lines have shown that the choice between NHEJ or HR pathways depends on cell cycle stage. Surprisingly, cell cycle regulation of DSB repair has not been examined in normal human cells with intact cell cycle checkpoints. Here we measured the efficiency of NHEJ and HR at different cell cycle stages in hTERT-immortalized diploid human fibroblasts. We utilized cells with chromosomally-integrated fluorescent reporter cassettes, in which a unique DSB is introduced by a rare-cutting endonuclease. We show that NHEJ is active throughout the cell cycle, and its activity increases as cells progress from G1 to G2/M (G1efficiency of NHEJ was higher than HR at all cell cycle stages. We conclude that human somatic cells utilize error-prone NHEJ as the major DSB repair pathway at all cell cycle stages, while HR is used, primarily, in the S phase. PMID:18769152

  2. Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Petra Langerak

    2011-09-01

    Full Text Available The multifunctional Mre11-Rad50-Nbs1 (MRN protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR repair factor to double-strand breaks (DSBs. HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA overhangs that bind Replication Protein A (RPA complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1 DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.

  3. ENERGY EFFICIENCY OF DIESEL LOCOMOTIVE HYDRAULIC TRANSMISSION TESTS AT LOCOMOTIVE REPAIR PLANT

    Directory of Open Access Journals (Sweden)

    B. E. Bodnar

    2015-10-01

    Full Text Available Purpose. In difficult economic conditions, cost reduction of electricity consumption for the needs of production is an urgent task for the country’s industrial enterprises. Technical specifications of enterprises, which repair diesel locomotive hydraulic transmission, recommend conducting a certain amount of evaluation and regulatory tests to monitor their condition after repair. Experience shows that a significant portion of hydraulic transmission defects is revealed by bench tests. The advantages of bench tests include the ability to detect defects after repair, ease of maintenance of the hydraulic transmission and relatively low labour intensity for eliminating defects. The quality of these tests results in the transmission resource and its efficiency. Improvement of the technology of plant post-repairs hydraulic tests in order to reduce electricity consumption while testing. Methodology. The possible options for hydraulic transmission test bench improvement were analysed. There was proposed an energy efficiency method for diesel locomotive hydraulic transmission testing in locomotive repair plant environment. This is achieved by installing additional drive motor which receives power from the load generator. Findings. Based on the conducted analysis the necessity of improving the plant stand testing of hydraulic transmission was proved. The variants of the stand modernization were examined. The test stand modernization analysis was conducted. Originality. The possibility of using electric power load generator to power the stand electric drive motor or the additional drive motor was theoretically substantiated. Practical value. A variant of hydraulic transmission test stand based on the mutual load method was proposed. Using this method increases the hydraulic transmission load range and power consumption by stand remains unchanged. The additional drive motor will increase the speed of the input shaft that in its turn wil allow testing in

  4. The Heterochromatic Barrier to DNA Double Strand Break Repair: How to Get the Entry Visa

    Directory of Open Access Journals (Sweden)

    Aaron A. Goodarzi

    2012-09-01

    Full Text Available Over recent decades, a deep understanding of pathways that repair DNA double strand breaks (DSB has been gained from biochemical, structural, biophysical and cellular studies. DNA non-homologous end-joining (NHEJ and homologous recombination (HR represent the two major DSB repair pathways, and both processes are now well understood. Recent work has demonstrated that the chromatin environment at a DSB significantly impacts upon DSB repair and that, moreover, dramatic modifications arise in the chromatin surrounding a DSB. Chromatin is broadly divided into open, transcriptionally active, euchromatin (EC and highly compacted, transcriptionally inert, heterochromatin (HC, although these represent extremes of a spectrum. The HC superstructure restricts both DSB repair and damage response signaling. Moreover, DSBs within HC (HC-DSBs are rapidly relocalized to the EC-HC interface. The damage response protein kinase, ataxia telangiectasia mutated (ATM, is required for HC-DSB repair but is dispensable for the relocalization of HC-DSBs. It has been proposed that ATM signaling enhances HC relaxation in the DSB vicinity and that this is a prerequisite for HC-DSB repair. Hence, ATM is essential for repair of HC-DSBs. Here, we discuss how HC impacts upon the response to DSBs and how ATM overcomes the barrier that HC poses to repair.

  5. Microhomology directs diverse DNA break repair pathways and chromosomal translocations.

    Directory of Open Access Journals (Sweden)

    Diana D Villarreal

    Full Text Available Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR events after a DNA double-strand break (DSB in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.

  6. Mechanisms of DNA repair and radio-induced mutagenesis in higher eukaryotes; Mecanismes de reparation et mutagenese radio-induite chez les eucaryotes superieurs

    Energy Technology Data Exchange (ETDEWEB)

    Averbeck, D. [Centre Universitaire d' Orsay, Institut Curie, Section de Recherche, Lab. Raymond-Latarjet, UMR 2027 CNRS, 91 (France)

    2000-10-01

    Cells of higher eukaryotes possess several very efficient systems for the repair of radiation-induced lesions in DNA. Different strategies have been adopted at the cellular level to remove or even tolerate various types of lesions in order to assure survival and limit the mutagenic consequences. In mammalian cells, the main DNA repair systems comprise direct reversion of damage, excision of damage and exchange mechanisms with intact DNA. Among these, the direct ligation of single strand breaks (SSB) by a DNA ligase and the multi-enzymatic repair systems of mismatch repair, base and nucleotide excision repair as well as the repair of double strand breaks (DSB) by homologous recombination or non homologous end-joining are the most important systems. Most of these processes are error-free except the non homologous end-joining pathway used for the repair of DSB. Moreover, certain lesions can be tolerated by more or less accurately acting polymerases capable of performing trans-lesion DNA syntheses. The DNA repair systems are intimately integrated in the network of cellular regulation. Some of their components are DNA damage inducible. Radiation-induced mutagenesis is largely due to unrepaired DNA damage but also involves error-prone repair processes like the repair of DSB by non-homologous end-joining. Generally, mammalian cells are well prepared to repair radiation-induced lesions. However, some questions remain to be asked about mechanistic details and efficiencies of the systems for removing certain types of radiation-damage and about their order and timing of action. The answers to these questions would be important for radioprotection as well as radiotherapy. (author)

  7. Double-strand break repair based on short-homology regions is suppressed under terminal deoxynucleotidyltransferase expression, as revealed by a novel vector system for analysing DNA repair by nonhomologous end joining.

    Science.gov (United States)

    Maezawa, So; Nakano, Saori; Kuniya, Takaaki; Koiwai, Osamu; Koiwai, Kotaro

    2016-01-01

    We have constructed a novel, nonhomologous end-joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I-SceI generated a double-strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing the repaired NAV lacked the ccdB gene which slows cell proliferation, the cultures were enriched in cells containing repaired DSBs. DNA sequence analysis of the DSB junctions indicated that the repair was carried out mainly by using the closest homology sequence. Use of the NAV yielded rapid results within 3 days after transfection. We then used the NAV to analyse NHEJ in cells overexpressing terminal deoxynucleotidyltransferase (TdT). The results indicated that TdT suppresses DNA repair that is based on short (one- or two-base) homology regions, to efficiently add deoxynucleotides during VDJ recombination in lymphoid cells.

  8. STUDY OF THE PARAMETERS OF EFFICIENCY IN CENTRES FOR REPAIR OF AGRICULTURAL MACHINERY

    OpenAIRE

    2015-01-01

    The paper makes a thorough study of the parameters of efficiency in the centers for the repair of agricultural machinery, considering production and technological structure, the basic principles for design of the process of service, the quantitative indicators for servicing. It presents a theoretical model for the management of services in the service business, taking into account the basic system requirements for maintenance of agricultural machinery, the main elements of the standards of cu...

  9. The involvement of human RECQL4 in DNA double-strand break repair

    DEFF Research Database (Denmark)

    Singh, Dharmendra Kumar; Karmakar, Parimal; Aamann, Maria Diget

    2010-01-01

    Rothmund-Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas....... The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double-strand break (DSB) repair. The results show that RECQL4-deficient fibroblasts are moderately...... sensitive to gamma-irradiation and accumulate more gammaH2AX and 53BP1 foci than control fibroblasts. This is suggestive of defects in efficient repair of DSB's in the RECQL4-deficient fibroblasts. Real time imaging of live cells using laser confocal microscopy shows that RECQL4 is recruited early to laser...

  10. Dominant negative mutant of Plasmodium Rad51 causes reduced parasite burden in host by abrogating DNA double-strand break repair.

    Science.gov (United States)

    Roy, Nabamita; Bhattacharyya, Sunanda; Chakrabarty, Swati; Laskar, Shyamasree; Babu, Somepalli Mastan; Bhattacharyya, Mrinal Kanti

    2014-10-01

    Malaria parasites survive through repairing a plethora of DNA double-stranded breaks (DSBs) experienced during their asexual growth. In Plasmodium Rad51 mediated homologous recombination (HR) mechanism and homology-independent alternative end-joining mechanism have been identified. Here we address whether loss of HR activity can be compensated by other DSB repair mechanisms. Creating a transgenic Plasmodium line defective in HR function, we demonstrate that HR is the most important DSB repair pathway in malarial parasite. Using mouse malaria model we have characterized the dominant negative effect of PfRad51(K143R) mutant on Plasmodium DSB repair and host-parasite interaction. Our work illustrates that Plasmodium berghei harbouring the mutant protein (PfRad51(K143R)) failed to repair DSBs as evidenced by hypersensitivity to DNA-damaging agent. Mice infected with mutant parasites lived significantly longer with markedly reduced parasite burden. To better understand the effect of mutant PfRad51(K143R) on HR, we used yeast as a surrogate model and established that the presence of PfRad51(K143R) completely inhibited DNA repair, gene conversion and gene targeting. Biochemical experiment confirmed that very low level of mutant protein was sufficient for complete disruption of wild-type PfRad51 activity. Hence our work provides evidence that HR pathway of Plasmodium could be efficiently targeted to curb malaria.

  11. [The correlations between aging of the human body, oxidative stress and reduced efficiency of repair systems].

    Science.gov (United States)

    Michalak, Aleksandra; Krzeszowiak, Jakub; Markiewicz-Górka, Iwona

    2014-12-15

    The article presents an current knowledge overview about the importance of oxidative stress and reduced efficiency of repair processes during the aging process of the human body. Oxidative damage to cellular macromolecules (proteins, lipids, nucleic acids), are formed under the influence of reactive oxygen species (ROS). They are the part of important mechanism which is responsible for the process of aging and the development of many diseases. The most important effects result from DNA damage, due to the mutations formation, which can lead to the development of tumors. However, a well-functioning repair systems (i.a. homologous recombination) remove the damage and prevent harmful changes in the cells. Lipid peroxidation products also cause oxidative modification of nucleic acids (and proteins). Proteins and fats also have repair systems, but much simpler than those responsible for the repair of nucleic acids. Unfortunately, with increasing age, they are more weakened, which contributes to increase numbers of cell damage, and consequently development of diseases specific to old age: cancer, neurodegenerative diseases or atherosclerosis.

  12. Comparative Sequence, Structure and Redox Analyses of Klebsiella pneumoniae DsbA Show That Anti-Virulence Target DsbA Enzymes Fall into Distinct Classes

    OpenAIRE

    Fabian Kurth; Kieran Rimmer; Lakshmanane Premkumar; Biswaranjan Mohanty; Wilko Duprez; Halili, Maria A; Shouldice, Stephen R.; Begoña Heras; Fairlie, David P.; Martin J. Scanlon; Jennifer L Martin

    2013-01-01

    Bacterial DsbA enzymes catalyze oxidative folding of virulence factors, and have been identified as targets for antivirulence drugs. However, DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA (EcDsbA). Nonetheless, sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors, highlighting the potential to inhibit virulenc...

  13. Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin

    OpenAIRE

    Kakarougkas, Andreas; Ismail, Amani; Chambers, Anna; Riballo, Queti; Herbert, Alex; Kunzel, Julia; Lobrich, Markus; Jeggo, Penny; Downs, Jessica

    2014-01-01

    Summary Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes ...

  14. The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair.

    Science.gov (United States)

    Robert, Flavie; Hardy, Sara; Nagy, Zita; Baldeyron, Céline; Murr, Rabih; Déry, Ugo; Masson, Jean-Yves; Papadopoulo, Dora; Herceg, Zdenko; Tora, Làszlò

    2006-01-01

    Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

  15. Defense Science Board (DSB) Summer Study Report on Strategic Surprise

    Science.gov (United States)

    2015-07-01

    DSB Summer Study Report on Strategic Surprise July 2015 Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden...SUBTITLE DSB Summer Study Report on Strategic Surprise 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT...NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Defense Science Board ( DSB ),The Pentagon ,OUSD(AT&L

  16. Structure of a DsbF homologue from Corynebacterium diphtheriae.

    Science.gov (United States)

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-09-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein.

  17. Diversity of the Epsilonproteobacteria Dsb (disulfide bond) systems.

    Science.gov (United States)

    Bocian-Ostrzycka, Katarzyna M; Grzeszczuk, Magdalena J; Dziewit, Lukasz; Jagusztyn-Krynicka, Elżbieta K

    2015-01-01

    The bacterial proteins of the Dsb family-important components of the post-translational protein modification system-catalyze the formation of disulfide bridges, a process that is crucial for protein structure stabilization and activity. Dsb systems play an essential role in the assembly of many virulence factors. Recent rapid advances in global analysis of bacteria have thrown light on the enormous diversity among bacterial Dsb systems. While the Escherichia coli disulfide bond-forming system is quite well understood, the mechanisms of action of Dsb systems in other bacteria, including members of class Epsilonproteobacteria that contain pathogenic and non-pathogenic bacteria colonizing extremely diverse ecological niches, are poorly characterized. Here we present a review of current knowledge on Epsilonproteobacteria Dsb systems. We have focused on the Dsb systems of Campylobacter spp. and Helicobacter spp. because our knowledge about Dsb proteins of Wolinella and Arcobacter spp. is still scarce and comes mainly from bioinformatic studies. Helicobacter pylori is a common human pathogen that colonizes the gastric epithelium of humans with severe consequences. Campylobacter spp. is a leading cause of zoonotic enteric bacterial infections in most developed and developing nations. We focus on various aspects of the diversity of the Dsb systems and their influence on pathogenicity, particularly because Dsb proteins are considered as potential targets for a new class of anti-virulence drugs to treat human infections by Campylobacter or Helicobacter spp.

  18. Diversity of the Epsilonproteobacteria Dsb (disulfide bond systems

    Directory of Open Access Journals (Sweden)

    Katarzyna Marta Bocian-Ostrzycka

    2015-06-01

    Full Text Available The bacterial proteins of the Dsb family – important components of the posttranslational protein modification system – catalyze the formation of disulfide bridges, a process that is crucial for protein structure stabilization and activity. Dsb systems play an essential role in the assembly of many virulence factors. Recent rapid advances in global analysis of bacteria have thrown light on the enormous diversity among bacterial Dsb systems. While the Escherichia coli disulfide bond-forming system is quite well understood, the mechanisms of action of Dsb systems in other bacteria, including members of class Epsilonproteobacteria that contain pathogenic and non-pathogenic bacteria colonizing extremely diverse ecological niches, are poorly characterized. Here we present a review of current knowledge on Epsilonproteobacteria Dsb systems. We have focused on the Dsb systems of Campylobacter spp. and Helicobacter spp. because our knowledge about Dsb proteins of Wolinella and Arcobacter spp. is still scarce and comes mainly from bioinformatic studies. Helicobacter pylori is a common human pathogen that colonizes the gastric epithelium of humans with severe consequences. Campylobacter spp. is a leading cause of zoonotic enteric bacterial infections in most developed and developing nations. We focus on various aspects of the diversity of the Dsb systems and their influence on pathogenicity, particularly because Dsb proteins are considered as potential targets for a new class of anti-virulence drugs to treat human infections by Campylobacter or Helicobacter spp.

  19. DNA Repair by Homologous Recombination, But Not by Nonhomologous End Joining, Is Elevated in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Zhiyong Mao

    2009-07-01

    Full Text Available Aberrant double-stranded break (DSB repair leads to genomic instability, which is a hallmark of malignant cells. Double-stranded breaks are repaired by two pathways: homologous recombination (HR and nonhomologous DNA end joining (NHEJ. It is not known whether these repair pathways are affected in sporadic breast tumors. Here, we examined the efficiency of HR and NHEJ repair in a panel of sporadic breast cancer cell lines and tested whether the efficiency of HR or NHEJ correlates with radioresistance. Homologous recombination and NHEJ in breast cancer cells were analyzed using in vivo fluorescent assays. Unexpectedly, our analysis revealed that the efficiency of HR is significantly elevated in breast cancer cells compared with normal mammary epithelial cells. In contrast, the efficiency of NHEJ in breast cancer cells is not different from normal cells. Overall, breast cancer cells were more sensitive to radiation than normal cells, but the levels of resistance did not correlate with either HR or NHEJ efficiency. Thus, we demonstrate that sporadic breast cancers are not associated with a deficiency in DSB repair, but rather with upregulation of the HR pathway. Our finding of elevated HR in sporadic breast cancer cell lines suggests that therapies directed against the components of HR will be highly tumor-specific.

  20. DNA Repair by Homologous Recombination, But Not by Nonhomologous End Joining, Is Elevated in Breast Cancer Cells12

    Science.gov (United States)

    Mao, Zhiyong; Jiang, Ying; Liu, Xiang; Seluanov, Andrei; Gorbunova, Vera

    2009-01-01

    Aberrant double-stranded break (DSB) repair leads to genomic instability, which is a hallmark of malignant cells. Double-stranded breaks are repaired by two pathways: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). It is not known whether these repair pathways are affected in sporadic breast tumors. Here, we examined the efficiency of HR and NHEJ repair in a panel of sporadic breast cancer cell lines and tested whether the efficiency of HR or NHEJ correlates with radioresistance. Homologous recombination and NHEJ in breast cancer cells were analyzed using in vivo fluorescent assays. Unexpectedly, our analysis revealed that the efficiency of HR is significantly elevated in breast cancer cells compared with normal mammary epithelial cells. In contrast, the efficiency of NHEJ in breast cancer cells is not different from normal cells. Overall, breast cancer cells were more sensitive to radiation than normal cells, but the levels of resistance did not correlate with either HR or NHEJ efficiency. Thus, we demonstrate that sporadic breast cancers are not associated with a deficiency in DSB repair, but rather with upregulation of the HR pathway. Our finding of elevated HR in sporadic breast cancer cell lines suggests that therapies directed against the components of HR will be highly tumor-specific. PMID:19568413

  1. Optimization of DsbA Purification from Recombinant Escherichia coil Broth Using Box-Behnken Design Methodology

    Institute of Scientific and Technical Information of China (English)

    LUO Man; GUAN Yixin; YAO Shanjing

    2013-01-01

    Disulfide bond formation protein A(DsbA)is one of the important helper proteins for folding in protein synthesis in vivo.In this study,purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method,a statistic-based design of experiments.The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem,which takes the protein recovery,purification efficiency and throughput of ion-exchange chromatography into account.After the optimization,protein recovery of 96.8% and purity higher than 95% DsbA was achieved,and the productivity was (377.9±1.7)mg soluble DsbA per liter broth.The purified protein was identified by peptide mass fingerprinting matching the record of gil2624856,a mutant of DsbA.The DsbA was preliminarily applied to the refolding of denatured lysozyme m vitro.

  2. A modified fluorimetric neutral filter elution method for analyzing radiation-induced double strand break and repair.

    Science.gov (United States)

    Goutham, Venkatesh H; Kamalesh, Mumbrekar D; Guruprasad, Parashiva K; Vadhiraja, Manjunath B; Satyamoorthy, Kapaettu; Rao Bola Satish, Sadashiva

    2011-07-15

    Neutral filter elution assay is one of the methods used for detection of DNA double strand breaks (DSBs). However, it is laborious, expensive, and hazardous (radiolabeled precursors for DSB detection and scintillation counter for quantification), making it a less preferred method for DSB detection. In the present study, an attempt was made to improve the existing neutral filter elution assay by making use of fluorescent dye (PicoGreen) and microfiltration assembly for eluting the fragmented DNA, thereby reducing the cost and time required for the assay. We studied the effect of dye dilution, pH conditions, and cell number as a part of method standardization. X-ray dose-response and repair kinetics in lymphocytes as well as cell lines were studied for validating the sensitivity of the assay. A linear dose-response relationship for DSBs was observed at a cell number of 4×10(5)cells, a dye dilution of 500-fold, and at pH 10. Repair kinetics revealed a time-dependent repair of DSBs up to 360 min of posttreatment, indicating its usefulness in DSB repair studies. In conclusion, the present modified method is more efficient (in terms of cell number), cost effective, less time-consuming, and less hazardous compared to the existing method. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

    Science.gov (United States)

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.

  4. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair

    Science.gov (United States)

    Svendsen, Jennifer M.; Smogorzewska, Agata; Sowa, Mathew E.; O’Connell, Brenda C.; Gygi, Steven P.; Elledge, Stephen J.; Harper, J. Wade

    2009-01-01

    Summary Structure-specific endonucleases mediate repair of DNA structures formed from replication fork collapse or during double-strand break (DSB) repair. Here we identify BTBD12 as the human ortholog of the budding yeast DNA repair factor Slx4p and D. melanogaster MUS312. Human SLX4 forms a multiprotein complex with the ERCC4(XPF)-ERCC1, MUS81-EME1, and SLX1 endonucleases, and also associates with MSH2/MSH3 mismatch repair complex, telomere binding complex TERF2(TRF2)-TERF2IP(RAP1), the protein kinase PLK1 and the uncharacterized protein C20orf94. Depletion of SLX4 causes sensitivity to mitomycin C and camptothecin, and reduces the efficiency of DSB repair in vivo. SLX4 complexes cleave 3’-flap, 5’-flap and replication fork structures; yet unlike other endonucleases associated with SLX4, the SLX1-SLX4 module promotes symmetrical cleavage of static and migrating Holliday junctions (HJs), identifying SLX1-SLX4 as a HJ resolvase. Thus, SLX4 assembles a modular tool-kit for repair of specific types of DNA lesions and is critical for cellular responses to replication fork failure. PMID:19596235

  5. Efficiency of olaparib in colorectal cancer patients with an alteration of the homologous repair protein.

    Science.gov (United States)

    Ghiringhelli, Francois; Richard, Corentin; Chevrier, Sandy; Végran, Frédérique; Boidot, Romain

    2016-12-28

    Precision medicine is defined by the administration of drugs based on the tumor's particular genetic characteristics. It is developing quickly in the field of cancer therapy. For example, KRAS, NRAS and BRAF genetic testing demonstrates its efficiency for precision medicine in colorectal cancer (CRC). Besides for these well-known mutations, the purpose of performing larger genetic testing in this pathology is unknown. Recent reports have shown that using the poly ADP ribose polymerase (PARP) inhibitor olaparib in patients with homologous repair enzyme deficiency gave positive clinical results in breast, ovarian and prostate cancers. We have reported here the cases of 2 patients with multi-treated metastatic CRC who underwent somatic and constitutional exome analyses. The analyses revealed a loss of function mutation in a homologous repair enzyme resulting in the loss of heterozygosity for both patients (Check2 for the first patient and RAD51C for the second one). Both patients were treated with off-label usage of olaparib. While the first patient showed clinical benefit, reduction of carcinoembryonic antigen tumor marker and radiologic response, the second patient quickly presented a progression of the tumor. Additional genetic analyses revealed a frameshift truncating mutation of the TP53BP1 gene in the patient who progressed. Interestingly, deficiency in TP53BP1 was previously described to confer resistance to olaparib in mice breast cancer models. Our findings suggest that exome analysis may be a helpful tool to highlight targetable mutations in CRC and that olaparib may be efficient in patients with a homologous repair deficiency.

  6. Expression and crystallization of DsbA from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Heras, B., E-mail: b.heras@imb.uq.edu.au; Kurz, M.; Jarrott, R.; Byriel, K. A.; Jones, A. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane QLD 4072 (Australia); Thöny-Meyer, L. [EMPA, Abteilung Biokompatible Werkstoffe, Lerchenfeldstrasse 5, CH-9014 St Gallen (Switzerland); Martin, J. L., E-mail: b.heras@imb.uq.edu.au [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane QLD 4072 (Australia)

    2007-11-01

    Free-interface diffusion crystallization chips were used to identify crystallization conditions for S. aureus DsbA, representing the first Gram-positive DsbA to be crystallized. Native and selenomethionine-derivative crystals diffracted to 2.1 and 2.4 Å resolution, respectively. Bacterial Dsb proteins catalyse the in vivo formation of disulfide bonds, a critical step in the stability and activity of many proteins. Most studies on Dsb proteins have focused on Gram-negative bacteria and thus the process of oxidative folding in Gram-positive bacteria is poorly understood. To help elucidate this process in Gram-positive bacteria, DsbA from Staphylococcus aureus (SaDsbA) has been focused on. Here, the expression, purification, crystallization and preliminary diffraction analysis of SaDsbA are reported. SaDsbA crystals diffract to a resolution limit of 2.1 Å and belong to the hexagonal space group P6{sub 5} or P6{sub 1}, with unit-cell parameters a = b = 72.1, c = 92.1 Å and one molecule in the asymmetric unit (64% solvent content)

  7. DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Wyrobek, Andrew J.

    2009-01-18

    Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

  8. Evidence that the pathway of disulfide bond formation in Escherichia coli involves interactions between the cysteines of DsbB and DsbA.

    OpenAIRE

    Guilhot, C; Jander, G.; Martin, N L; Beckwith, J

    1995-01-01

    Disulfide bond formation is catalyzed in the periplasm of Escherichia coli. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. (Kishigami et al. [Kishigami, S., Kanaya, E., Kikuchi, M. & Ito, K. (1995) J. Biol. ...

  9. Meiotic DSB patterning: A multifaceted process.

    Science.gov (United States)

    Cooper, Tim J; Garcia, Valerie; Neale, Matthew J

    2016-01-01

    Meiosis is a specialized two-step cell division responsible for genome haploidization and the generation of genetic diversity during gametogenesis. An integral and distinctive feature of the meiotic program is the evolutionarily conserved initiation of homologous recombination (HR) by the developmentally programmed induction of DNA double-strand breaks (DSBs). The inherently dangerous but essential act of DSB formation is subject to multiple forms of stringent and self-corrective regulation that collectively ensure fruitful and appropriate levels of genetic exchange without risk to cellular survival. Within this article we focus upon an emerging element of this control--spatial regulation--detailing recent advances made in understanding how DSBs are evenly distributed across the genome, and present a unified view of the underlying patterning mechanisms employed.

  10. Efficient Stagnation Avoidance for Manets with Local Repair Strategy Using ant Colony Optimization

    Directory of Open Access Journals (Sweden)

    Sharvani G S

    2012-10-01

    Full Text Available Wireless networks such as Mobile AdHoc Networks (MANETs have many advantages compared to wired networks. In MANETs the communication is not limited to a certain geometrical region. Swarm Intelligence based ACO algorithms provide interesting solutions to network routing problems. ACO based routing in MANETs will enhance the reliability and efficient packet delivery. They help in reducing control overhead due to their inherent scalable feature. The similarity between ant and nodes, colony and Wireless network helps to use ACO based routing in MANETs. The Termite Algorithms contains several tunable parameters and methods to automate the selection of optimal routes for different network conditions. However, Termite doesn’t contain methods for determination of QoS, Route Maintenance; Load balancing etc. The present work focuses on development of an efficient routing algorithm “Modified Termite algorithms” (MTA for MANETs. The MTA developed by adopting efficient pheromone evaporation technique will address to load balancing problems. By including QoS, efficient route maintenance, local repair strategy by prediction of node failures, the MTA is expected to enhance the performance of the network in terms of throughput, and reduction of End-to-end delay and routing overheads. The results of the analysis are presented in the paper.

  11. SCAI promotes DNA double-strand break repair in distinct chromosomal contexts

    DEFF Research Database (Denmark)

    Hansen, Rebecca Kring; Mund, Andreas; Poulsen, Sara Lund;

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer...... cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1......-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs...

  12. Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage.

    Science.gov (United States)

    Vu, Giang T H; Cao, Hieu X; Reiss, Bernd; Schubert, Ingo

    2017-02-28

    In order to prevent genome instability, cells need to be protected by a number of repair mechanisms, including DNA double-strand break (DSB) repair. The extent to which DSB repair, biased towards deletions or insertions, contributes to evolutionary diversification of genome size is still under debate. We analyzed mutation spectra in Arabidopsis thaliana and in barley (Hordeum vulgare) by PacBio sequencing of three DSB-targeted loci each, uncovering repair via gene conversion, single strand annealing (SSA) or nonhomologous end-joining (NHEJ). Furthermore, phylogenomic comparisons between A. thaliana and two related species were used to detect naturally occurring deletions during Arabidopsis evolution. Arabidopsis thaliana revealed significantly more and larger deletions after DSB repair than barley, and barley displayed more and larger insertions. Arabidopsis displayed a clear net loss of DNA after DSB repair, mainly via SSA and NHEJ. Barley revealed a very weak net loss of DNA, apparently due to less active break-end resection and easier copying of template sequences into breaks. Comparative phylogenomics revealed several footprints of SSA in the A. thaliana genome. Quantitative assessment of DNA gain and loss through DSB repair processes suggests deletion-biased DSB repair causing ongoing genome shrinking in A. thaliana, whereas genome size in barley remains nearly constant.

  13. Increased repair of {gamma}-induced DNA double-strand breaks at lower dose-rate in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Boucher, D.; Hindo, J.; Averbeck, D. [Centre Universitaire d' Orsay, Inst. Curie-Section de Recherche, Orsay CEDEX (France)]. E-mail: dietrich.averbeck@curie.u-psud.fr

    2004-02-01

    DNA double-strand breaks (DSBs) are highly cell damaging. We asked whether for a given dose a longer irradiation time would be advantageous for the repair of DSBs. Varying the {gamma}-irradiation dose and its delivery time (0.05 Gy/min low dose-rate (LDR) compared with 3.5 Gy/min high dose-rate), confluent Chinese hamster ovary cells (CHO-K1) and Ku80 mutant cells (xrs-6) deficient in nonhomologous end-joining (NHEJ) were irradiated in agarose plugs at room temperature using a cesium-137 {gamma}-ray source. We used pulsed-field gel electrophoresis (PFGE) to measure DSBs in terms of the fraction of activity released (FAR). At LDR, one third of DSBs were repaired in CHO-K1 but not in xrs-6 cells, indicating the involvement of NHEJ in the repair of {gamma}-induced DSBs at a prolonged irradiation incubation time. To improve DSB measurements, we introduced in our PFGE protocol an antioxidant at the cell lysis step, thus avoiding free-radical side reactions on DNA and spurious DSBs. Addition of the metal chelator deferoxamine (DFO) decreased more efficiently the basal DSB level than did reduced glutathione (GSH), showing that measuring DSBs in the absence of DFO reduces precision and underestimates the role of NHEJ in the dose-rate effect on DSB yield. (author)

  14. Use efficiency of dynamic stabilizer in the post-repair period of railways in Ukraine

    Directory of Open Access Journals (Sweden)

    O.V. Gubar

    2013-08-01

    Full Text Available Purpose. To analyze the interaction forces of the track and rolling stock at application of dynamic stabilizers, as well as the determination of the area, on which the track stabilization is necessary not only according to the technical indices but also according to the economic ones. Methodology.To achieve the research purpose the methods of analysis of material flows, peculiar to the places that need applications of dynamic stabilizers on railway transport tracks are used. Findings. Researches of relatively economic efficiency of dynamic stabilizers application, which were conducted by scientists during the last years, do not spread to comparison of cost of works on the track stabilizing and losses of railway for period of running. During the running period the trains move with a limited speed that causes corresponding financial losses. Speed limitation result in the both the motion time increase and in some occasions in the heavy consumption of fuel and energy resources. The more intensive track discord and expenses increase for its maintenance are observed in the sections of braking and acceleration immediately in front of and after the areas of limitation. The methodology relative to the estimation of economic efficiency of dynamic stabilizers application after completion of track repairs for the areas of Ukrainian railways with different operational conditions was developed. This methodology includes the losses calculation of railroad, which are predefined by extra motion time, heavy consumption of fuel and energy resources and charges for current maintenance. Originality. The methodology of cost effectiveness evaluating of the dynamic stabilizers application in the post-repair period was developed. Such an approach would allow one to take the rational decisions taking into account the features of the track sections, which were reconstructed. Practical value. The obtained results will rationally assign the works on the dynamic track

  15. Exo1 and Mre11 execute meiotic DSB end resection in the protist Tetrahymena.

    Science.gov (United States)

    Lukaszewicz, Agnieszka; Shodhan, Anura; Loidl, Josef

    2015-11-01

    The resection of 5'-DNA ends at a double-strand break (DSB) is an essential step in recombinational repair, as it exposes 3' single-stranded DNA (ssDNA) tails for interaction with a repair template. In mitosis, Exo1 and Sgs1 have a conserved function in the formation of long ssDNA tails, whereas this step in the processing of programmed meiotic DSBs is less well-characterized across model organisms. In budding yeast, which has been most intensely studied in this respect, Exo1 is a major meiotic nuclease. In addition, it exerts a nuclease-independent function later in meiosis in the conversion of DNA joint molecules into ZMM-dependent crossovers. In order to gain insight into the diverse meiotic roles of Exo1, we investigated the effect of Exo1 deletion in the ciliated protist Tetrahymena. We found that Exo1 together with Mre11, but without the help of Sgs1, promotes meiotic DSB end resection. Resection is completely eliminated only if both Mre11 and Exo1 are missing. This is consistent with the yeast model where Mre11 promotes resection in the 3'-5' direction and Exo1 in the opposite 5'-3' direction. However, while the endonuclease activity of Mre11 is essential to create an entry site for exonucleases and hence to start resection in budding yeast, Tetrahymena Exo1 is able to create single-stranded DNA in the absence of Mre11. Excluding a possible contribution of the Mre11 cofactor Sae2 (Com1) as an autonomous endonuclease, we conclude that there exists another unknown nuclease that initiates DSB processing in Tetrahymena. Consistent with the absence of the ZMM crossover pathway in Tetrahymena, crossover formation is independent of Exo1.

  16. Rtt107 phosphorylation promotes localisation to DNA double-stranded breaks (DSBs and recombinational repair between sister chromatids.

    Directory of Open Access Journals (Sweden)

    Pranav Ullal

    Full Text Available Efficient repair of DNA double-stranded breaks (DSB requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5-Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR and propose that its recruitment to DSBs, together with the Smc5-Smc6 complex is important for repair through the SCR pathway.

  17. Simulation of DSB yield for high LET radiation.

    Science.gov (United States)

    Friedrich, T; Durante, M; Scholz, M

    2015-09-01

    A simulation approach for the calculation of the LET-dependent yield of double-strand breaks (DSB) is presented. The model considers DSB formed as two close-lying single-strand breaks (SSB), whose formation is mediated by both intra-track processes (single electrons) or at local doses larger than about 1000 Gy in particle tracks also by electron inter-track processes (two independent electron tracks). A Monte Carlo algorithm and an analytical formula for the DSB yield are presented. The approach predicts that the DSB yield is enhanced after charged particle irradiation of high LET compared with X-ray or gamma radiation. It is used as an inherent part of the local effect model, which is applied to estimate the relative biological effectiveness of high LET radiation.

  18. Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

    Science.gov (United States)

    Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-01

    The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

  19. DNA double strand break (DSB) induction and cell survival in iodine-enhanced computed tomography (CT)

    Science.gov (United States)

    Streitmatter, Seth W.; Stewart, Robert D.; Jenkins, Peter A.; Jevremovic, Tatjana

    2017-08-01

    A multi-scale Monte Carlo model is proposed to assess the dosimetric and biological impact of iodine-based contrast agents commonly used in computed tomography. As presented, the model integrates the general purpose MCNP6 code system for larger-scale radiation transport and dose assessment with the Monte Carlo damage simulation to determine the sub-cellular characteristics and spatial distribution of initial DNA damage. The repair-misrepair-fixation model is then used to relate DNA double strand break (DSB) induction to reproductive cell death. Comparisons of measured and modeled changes in reproductive cell survival for ultrasoft characteristic k-shell x-rays (0.25-4.55 keV) up to orthovoltage (200-500 kVp) x-rays indicate that the relative biological effectiveness (RBE) for DSB induction is within a few percent of the RBE for cell survival. Because of the very short range of secondary electrons produced by low energy x-ray interactions with contrast agents, the concentration and subcellular distribution of iodine within and near cellular targets have a significant impact on the estimated absorbed dose and number of DSB produced in the cell nucleus. For some plausible models of the cell-level distribution of contrast agent, the model predicts an increase in RBE-weighted dose (RWD) for the endpoint of DSB induction of 1.22-1.40 for a 5-10 mg ml-1 iodine concentration in blood compared to an RWD increase of 1.07  ±  0.19 from a recent clinical trial. The modeled RWD of 2.58  ±  0.03 is also in good agreement with the measured RWD of 2.3  ±  0.5 for an iodine concentration of 50 mg ml-1 relative to no iodine. The good agreement between modeled and measured DSB and cell survival estimates provides some confidence that the presented model can be used to accurately assess biological dose for other concentrations of the same or different contrast agents.

  20. Why is DsbA such an oxidizing disulfide catalyst?

    DEFF Research Database (Denmark)

    Grauschopf, U; Winther, Jakob R.; Korber, P;

    1995-01-01

    DsbA, a member of the thioredoxin family of disulfide oxidoreductases, acts in catalyzing disulfide bond formation by donating its disulfide to newly translocated proteins. We have found that the two central residues within the active site Cys-30-Pro-31-His-32-Cys-33 motif are critical in determi......DsbA, a member of the thioredoxin family of disulfide oxidoreductases, acts in catalyzing disulfide bond formation by donating its disulfide to newly translocated proteins. We have found that the two central residues within the active site Cys-30-Pro-31-His-32-Cys-33 motif are critical...... in determining the exceptional oxidizing power of DsbA. Mutations that change these two residues can alter the equilibrium oxidation potential of DsbA by more than 1000-fold. A quantitative explanation for the very high redox potential of DsbA was found by measuring the pKa of a single residue, Cys-30. The p......Ka of Cys-30 varied dramatically from mutant to mutant and could accurately predict the oxidizing power of each DsbA mutant protein....

  1. Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways.

    Science.gov (United States)

    Sears, Catherine R; Turchi, John J

    2012-07-13

    The treatment for advanced stage non-small cell lung cancer (NSCLC) often includes platinum-based chemotherapy and IR. Cisplatin and IR combination therapy display schedule and dose-dependent synergy, the mechanism of which is not completely understood. In a series of in vitro and cell culture assays in a NSCLC model, we investigated both the downstream and direct treatment and damage effects of cisplatin on NHEJ catalyzed repair of a DNA DSB. The results demonstrate that extracts prepared from cisplatin-treated cells are fully capable of NHEJ catalyzed repair of a DSB using a non-cisplatin-damaged DNA substrate in vitro. Similarly, using two different host cell reactivation assays, treatment of cells prior to transfection of a linear, undamaged reporter plasmid revealed no reduction in NHEJ compared with untreated cells. In contrast, transfection of a linear GFP-reporter plasmid containing site-specific, cisplatin lesions 6-bp from the termini revealed a significant impairment in DSB repair of the cisplatin-damaged DNA substrates in the absence of cellular treatment with cisplatin. Together, these data demonstrate that impaired NHEJ in combined cisplatin-IR treated cells is likely the result of a direct effect of cisplatin-DNA lesions near a DSB and that the indirect cellular effects of cisplatin treatment are not significant contributors to the synergistic cytotoxicity observed with combination cisplatin-IR treatment.

  2. Mutants in DsbB that appear to Redirect Oxidation Through the Disulfide Isomerization Pathway

    OpenAIRE

    Pan, Jonathan L.; Sliskovic, Inga; Bardwell, James C. A.

    2008-01-01

    Disulfide bond formation occurs in secreted proteins in Escherichia coli when the disulfide oxidoreductase DsbA, a soluble periplasmic protein, nonspecifically transfers a disulfide to a substrate protein. The catalytic disulfide of DsbA is regenerated by the inner membrane protein DsbB. To help identify the specificity determinants in DsbB and to understand the nature of the kinetic barrier preventing direct oxidation of newly secreted proteins by DsbB, we imposed selective pressure to find ...

  3. Spectrum of Radiation-Induced Clustered Non-DSB Damage - A Monte Carlo Track Structure Modeling and Calculations.

    Science.gov (United States)

    Watanabe, Ritsuko; Rahmanian, Shirin; Nikjoo, Hooshang

    2015-05-01

    The aim of this report is to present the spectrum of initial radiation-induced cellular DNA damage [with particular focus on non-double-strand break (DSB) damage] generated by computer simulations. The radiation types modeled in this study were monoenergetic electrons (100 eV-1.5 keV), ultrasoft X-ray photons Ck, AlK and TiK, as well as some selected ions including 3.2 MeV/u proton; 0.74 and 2.4 MeV/u helium ions; 29 MeV/u nitrogen ions and 950 MeV/u iron ions. Monte Carlo track structure methods were used to simulate damage induction by these radiation types in a cell-mimetic condition from a single-track action. The simulations took into account the action of direct energy deposition events and the reaction of hydroxyl radicals on atomistic linear B-DNA segments of a few helical turns including the water of hydration. Our results permitted the following conclusions: a. The absolute levels of different types of damage [base damage, simple and complex single-strand breaks (SSBs) and DSBs] vary depending on the radiation type; b. Within each damage class, the relative proportions of simple and complex damage vary with radiation type, the latter being higher with high-LET radiations; c. Overall, for both low- and high-LET radiations, the ratios of the yields of base damage to SSBs are similar, being about 3.0 ± 0.2; d. Base damage contributes more to the complexity of both SSBs and DSBs, than additional SSB damage and this is true for both low- and high-LET radiations; and e. The average SSB/DSB ratio for low-LET radiations is about 18, which is about 5 times higher than that for high-LET radiations. The hypothesis that clustered DNA damage is more difficult for cells to repair has gained currency among radiobiologists. However, as yet, there is no direct in vivo experimental method to validate the dependence of kinetics of DNA repair on DNA damage complexity (both DSB and non-DSB types). The data on the detailed spectrum of DNA damage presented here, in particular

  4. Identification and Functional Analysis of an Immunoreactive DsbA-Like Thio-Disulfide Oxidoreductase of Ehrlichia spp.

    OpenAIRE

    McBride, Jere W.; Ndip, Lucy M.; Vsevolod L Popov; David H Walker

    2002-01-01

    Novel homologous DsbA-like disulfide bond formation (Dsb) proteins of Ehrlichia chaffeensis and Ehrlichia canis were identified which restored DsbA activity in complemented Escherichia coli dsbA mutants. Recombinant Ehrlichia Dsb (eDsb) proteins were recognized by sera from E. canis-infected dogs but not from E. chaffeensis-infected patients. The eDsb proteins were observed primarily in the periplasm of E. chaffeensis and E. canis.

  5. Repair Pathway Choices and Consequences at the Double-Strand Break.

    Science.gov (United States)

    Ceccaldi, Raphael; Rondinelli, Beatrice; D'Andrea, Alan D

    2016-01-01

    DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including genomic instability and cell death. Indeed, misrepair of DSBs can lead to inappropriate end-joining events, which commonly underlie oncogenic transformation due to chromosomal translocations. Typically, cells employ two main mechanisms to repair DSBs: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). In addition, alternative error-prone DSB repair pathways, namely alternative end joining (alt-EJ) and single-strand annealing (SSA), have been recently shown to operate in many different conditions and to contribute to genome rearrangements and oncogenic transformation. Here, we review the mechanisms regulating DSB repair pathway choice, together with the potential interconnections between HR and the annealing-dependent error-prone DSB repair pathways.

  6. Role of ubiquitination in meiotic recombination repair

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associated with ubiquitination with regard to homologous recombination (HR)-dependent DSB repair.

  7. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

    Science.gov (United States)

    Hüttner, Clemens; Murauer, Eva M.; Hainzl, Stefan; Kocher, Thomas; Neumayer, Anna; Reichelt, Julia; Bauer, Johann W.; Koller, Ulrich

    2016-01-01

    RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders. PMID:27669223

  8. Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining.

    Science.gov (United States)

    Jessulat, Matthew; Malty, Ramy H; Nguyen-Tran, Diem-Hang; Deineko, Viktor; Aoki, Hiroyuki; Vlasblom, James; Omidi, Katayoun; Jin, Ke; Minic, Zoran; Hooshyar, Mohsen; Burnside, Daniel; Samanfar, Bahram; Phanse, Sadhna; Freywald, Tanya; Prasad, Bhanu; Zhang, Zhaolei; Vizeacoumar, Franco; Krogan, Nevan J; Freywald, Andrew; Golshani, Ashkan; Babu, Mohan

    2015-07-01

    The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. miR-3940-5p enhances homologous recombination after DSB in Cr(VI) exposed 16HBE cell.

    Science.gov (United States)

    Li, Yang; Hu, Guiping; Li, Ping; Tang, Shichuan; Zhang, Ji; Jia, Guang

    2016-02-17

    Hexavalent chromium (Cr(VI)) is a well-recognized human carcinogen, yet the molecular mechanisms by which cause human cancer are still not well understood. MicroRNAs (miRNAs), which are small non-coding RNAs, are involved in carcinogenesis and DNA damage repair. Previous occupational population study showed that hexavalent chromium (Cr(VI)) downregulated plasma miR-3940-5p level, and a low miR-3940-5p level was associated with high XRCC2 expression in lymphocytes, indicating that miR-3940-5p maybe play a protective effect in Cr(VI) induced DNA damage. Here we investigated miR-3940-5p expression and its roles in DNA repair in Cr(VI)-treated 16HBE cells. miR-3940-5p change was detected by qRT-PCR. Rad51 foci formation and double strand break (DSB) were investigated to assess homologous recombination repair (HR) capacity by Immunofluorescent assay and Neutral Comet assay. XRCC2 expression was also evaluated after miRNA oligonucleotides transfection using Western blot. Cr(VI) treatment suppressed miR-3940-5p level in 16HBE cells. miR-3904-5p mimic downregulated XRCC2 expression. As a result, the formation of Rad51-foci was inhibited and DSB repair was prolonged. The results indicate that miR-3940-5p plays a protective effect in Cr(VI) induced DNA damage.

  10. Mystery of DNA repair: the role of the MRN complex and ATM kinase in DNA damage repair.

    Science.gov (United States)

    Czornak, Kamila; Chughtai, Sanaullah; Chrzanowska, Krystyna H

    2008-01-01

    Genomes are subject to a number of exogenous or endogenous DNA-damaging agents that cause DNA double-strand breaks (DSBs). These critical DNA lesions can result in cell death or a wide variety of genetic alterations, including deletions, translocations, loss of heterozygosity, chromosome loss, or chromosome fusions, which enhance genome instability and can trigger carcinogenesis. The cells have developed an efficient mechanism to cope with DNA damages by evolving the DNA repair machinery. There are 2 major DSB repair mechanisms: nonhomologous end joining (NHEJ) and homologous recombination (HR). One element of the repair machinery is the MRN complex, consisting of MRE11, RAD50 and NBN (previously described as NBS1), which is involved in DNA replication, DNA repair, and signaling to the cell cycle checkpoints. A number of kinases, like ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad-3-related), and DNA PKcs (DNA protein kinase catalytic subunit), phosphorylate various protein targets in order to repair the damage. If the damage cannot be repaired, they direct the cell to apoptosis. The MRN complex as well as repair kinases are also involved in telomere maintenance and genome stability. The dysfunction of particular elements involved in the repair mechanisms leads to genome instability disorders, like ataxia telangiectasia (A-T), A-T-like disorder (ATLD) and Nijmegen breakage syndrome (NBS). The mutated genes responsible for these disorders code for proteins that play key roles in the process of DNA repair. Here we present a detailed review of current knowledge on the MRN complex, kinases engaged in DNA repair, and genome instability disorders.

  11. Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Oorschot, Bregje van, E-mail: b.vanoorschot@amc.uva.nl [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Hovingh, Suzanne E. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Moerland, Perry D. [Bioinformatics Laboratory, Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Medema, Jan Paul; Stalpers, Lukas J.A. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Franken, Nicolaas A.P. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands)

    2014-03-01

    Purpose: To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Methods and Materials: Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Results: Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Conclusions: Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

  12. DNA-damage foci to detect and characterize DNA repair alterations in children treated for pediatric malignancies.

    Directory of Open Access Journals (Sweden)

    Nadine Schuler

    Full Text Available PURPOSE: In children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor. METHODS AND MATERIALS: In children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy. RESULTS: Between tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells. CONCLUSIONS: Analyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies.

  13. Cyclobutane pyrimidine dimers photolyase from extremophilic microalga: Remarkable UVB resistance and efficient DNA damage repair

    Energy Technology Data Exchange (ETDEWEB)

    Li, Chongjie [Key Laboratory of Marine Bioactive Substance, The First Institute of Oceanography, State Oceanic Administration, Qingdao 266061 (China); Ma, Li [Key Laboratory of Biofuels, and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101 (China); Mou, Shanli [Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao (China); Wang, Yibin, E-mail: wangyibin@fio.org.cn [Key Laboratory of Marine Bioactive Substance, The First Institute of Oceanography, State Oceanic Administration, Qingdao 266061 (China); Zheng, Zhou; Liu, Fangming; Qi, Xiaoqing; An, Meiling; Chen, Hao [Key Laboratory of Marine Bioactive Substance, The First Institute of Oceanography, State Oceanic Administration, Qingdao 266061 (China); Miao, Jinlai, E-mail: miaojinlai@163.com [Key Laboratory of Marine Bioactive Substance, The First Institute of Oceanography, State Oceanic Administration, Qingdao 266061 (China); State Key Laboratory of Biological Fermentation Engineering of Beer (In Preparation), Qingdao (China)

    2015-03-15

    Highlights: • Chlamydomonas sp. ICE-L photolyase gene PHR2 is first cloned and expressed in E. coli. • PHR2 complemented E. coli could efficiently survival from UV radiation. • Expressed PHR2 photolyase has distinct photo-reactivation activity in vitro. - Abstract: Bacteria living in the Antarctic region have developed several adaptive features for growth and survival under extreme conditions. Chlamydomonas sp. ICE-Lis well adapted to high levels of solar UV radiation. A putative photolyase was identified in the Chlamydomonas sp. ICE-L transcriptome. The complete cDNA sequence was obtained by RACE-PCR. This PHR encoding includes a polypeptide of 579 amino acids with clear photolyase signatures belonging to class II CPD-photolyases, sharing a high degree of homology with Chlamydomonas reinhardtii (68%). Real-time PCR was performed to investigate the potential DNA damage and responses following UVB exposure. CPD photolyase mRNA expression level increased over 50-fold in response to UVB radiation for 6 h. Using photolyase complementation assay, we demonstrated that DNA photolyase increased photo-repair more than 116-fold in Escherichia coli strain SY2 under 100 μw/cm{sup 2} UVB radiation. To determine whether photolyase is active in vitro, CPD photolyase was over-expressed. It was shown that pyrimidine dimers were split by the action of PHR2. This study reports the unique structure and high activity of the enzyme. These findings are relevant for further understanding of molecular mechanisms of photo-reactivation, and will accelerate the utilization of photolyase in the medical field.

  14. Dynamics and Mechanism of Efficient DNA Repair Reviewed by Active-Site Mutants

    Science.gov (United States)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2010-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA via a photoreaction which includes a series of light-driven electron transfers between the two-electron-reduced flavin cofactor FADH^- and the dimer. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of several active-site residues. With femtosecond resolution, we observed the significant change in the forward electron transfer from the excited FADH^- to the dimer and the back electron transfer from the repaired thymines by mutation of E274A, R226A, R342A, N378S and N378C. We also found that the mutation of E274A accelerates the bond-breaking of the thymine dimer. The dynamics changes are consistent with the quantum yield study of these mutants. These results suggest that the active-site residues play a significant role, structurally and chemically, in the DNA repair photocycle.

  15. Folding of DsbB in mixed micelles

    DEFF Research Database (Denmark)

    Otzen, Daniel

    2003-01-01

    is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a kinetic analysis of the folding of a membrane protein, disulfide bond reducing protein B (DsbB), in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate (SDS...... data are always open to alternative interpretations, time-resolved studies in mixed micelles provide a useful approach to measure membrane protein stability over a wide range of concentrations of SDS and DM, as well as a framework for the future characterization of the DsbB folding mechanism....

  16. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase.

    Science.gov (United States)

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with many DNA damage response proteins and form a unique chromatin structure known as the XY body. Interestingly, associated with the prolonged DSB repair, transcription is repressed in the XY body but not in autosomes, a phenomenon known as meiotic sex chromosome inactivation (MSCI), which is critical for male meiosis. Here using mice as model organisms, we briefly summarize recent progress on DSB repair in meiotic prophase and focus on the mechanism and function of DNA damage response in the XY body.

  17. Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint.

    Directory of Open Access Journals (Sweden)

    Martha Klovstad

    2008-02-01

    Full Text Available Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.

  18. Nucleotide excision repair is associated with the replisome and its efficiency depends on a direct interaction between XPA and PCNA.

    Directory of Open Access Journals (Sweden)

    Karin M Gilljam

    Full Text Available Proliferating cell nuclear antigen (PCNA is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM. The xeroderma pigmentosum group A (XPA protein, with a central but somewhat elusive role in nucleotide excision repair (NER, contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA(-/- cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6-4 photoproducts, and are consequently more arrested in S phase as compared to XPA(-/- cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.

  19. SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner

    Science.gov (United States)

    Xu, Zhu; Zhang, Lei; Zhang, Wenjun; Meng, Du; Zhang, Hongxia; Jiang, Ying; Xu, Xiaojun; Van Meter, Michael; Seluanov, Andrei; Gorbunova, Vera; Mao, Zhiyong

    2015-01-01

    In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER. PMID:25607651

  20. Applied Railway Optimization in Production Planning at DSB-S-tog - Tasks, Tools and Challenges

    DEFF Research Database (Denmark)

    Clausen, Jens

    2007-01-01

    Efficient public transportation is becoming increasingly vital for modern capitals. DSB S-tog a/s is the major supplier of rail traffic on the infrastructure of the city-rail network in Copenhagen. S-tog has experienced a demand for increasing volume and quality of the transportation offered....... In addition we describe on-going efforts in using mathematical models in activities such as timetable design and work-force planning. We also identify some organizatorial key factors, which have paved the way for extended use of optimization methods in railway production planning....

  1. CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle

    OpenAIRE

    Yun, Maximina H.; Hiom, Kevin

    2009-01-01

    The repair of DNA double-strand breaks (DSB) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSB occurs through non-homologous end-joining (NHEJ) or microhomology-mediated end-joining (MMEJ)1. These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional2, there is an increase in repair of DSB by homologous recomb...

  2. Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining.

    Science.gov (United States)

    Grabarz, Anastazja; Barascu, Aurélia; Guirouilh-Barbat, Josée; Lopez, Bernard S

    2012-01-01

    A DNA double strand break (DSB) is a highly toxic lesion, which can generate genetic instability and profound genome rearrangements. However, DSBs are required to generate diversity during physiological processes such as meiosis or the establishment of the immune repertoire. Thus, the precise regulation of a complex network of processes is necessary for the maintenance of genomic stability, allowing genetic diversity but protecting against genetic instability and its consequences on oncogenesis. Two main strategies are employed for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by single-stranded DNA (ssDNA) resection and requires sequence homology with an intact partner, while NHEJ requires neither resection at initiation nor a homologous partner. Thus, resection is an pivotal step at DSB repair initiation, driving the choice of the DSB repair pathway employed. However, an alternative end-joining (A-EJ) pathway, which is highly mutagenic, has recently been described; A-EJ is initiated by ssDNA resection but does not require a homologous partner. The choice of the appropriate DSB repair system, for instance according the cell cycle stage, is essential for genome stability maintenance. In this context, controlling the initial events of DSB repair is thus an essential step that may be irreversible, and the wrong decision should lead to dramatic consequences. Here, we first present the main DSB repair mechanisms and then discuss the importance of the choice of the appropriate DSB repair pathway according to the cell cycle phase. In a third section, we present the early steps of DSB repair i.e., DSB signaling, chromatin remodeling, and the regulation of ssDNA resection. In the last part, we discuss the competition between the different DSB repair mechanisms. Finally, we conclude with the importance of the fine tuning of this network for genome stability maintenance and for tumor protection in fine.

  3. Double-strand break repair on sex chromosomes: challenges during male meiotic prophase

    OpenAIRE

    Lu, Lin-Yu; Yu, Xiaochun

    2015-01-01

    During meiotic prophase, DNA double-strand break (DSB) repair-mediated homologous recombination (HR) occurs for exchange of genetic information between homologous chromosomes. Unlike autosomes or female sex chromosomes, human male sex chromosomes X and Y share little homology. Although DSBs are generated throughout male sex chromosomes, homologous recombination does not occur for most regions and DSB repair process is significantly prolonged. As a result, male sex chromosomes are coated with ...

  4. A new light on the meiotic DSB catalytic complex.

    Science.gov (United States)

    Robert, Thomas; Vrielynck, Nathalie; Mézard, Christine; de Massy, Bernard; Grelon, Mathilde

    2016-06-01

    Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs). More than 15 years ago, Spo11 was identified as the protein responsible for meiotic DSB formation, notably because of its striking similarities with the A subunit of topoisomerase VI (TopoVI). TopoVI are enzymes that modify DNA topology by generating transient DSBs and are active as heterotetramers, composed of two A and two B subunits. A2 dimers catalyse the DNA cleavage reaction, whereas the B subunits regulate A2 conformation, DNA capture, cleavage and re-ligation. The recent identification in plants and mammals of a B-like TopoVI subunit that interacts with SPO11 and is required for meiotic DSB formation makes us to reconsider our understanding of the meiotic DSB catalytic complex. We provide here an overview of the knowledge on TopoVI structure and mode of action and we compare them with their meiotic counterparts. This allows us to discuss the nature, structure and functions of the meiotic TopoVI-like complex during meiotic DSB formation.

  5. ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1.

    Science.gov (United States)

    Kijas, Amanda W; Lim, Yi Chieh; Bolderson, Emma; Cerosaletti, Karen; Gatei, Magtouf; Jakob, Burkhard; Tobias, Frank; Taucher-Scholz, Gisela; Gueven, Nuri; Oakley, Greg; Concannon, Patrick; Wolvetang, Ernst; Khanna, Kum Kum; Wiesmüller, Lisa; Lavin, Martin F

    2015-09-30

    The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

  6. Analysis of DNA double-strand break repair pathways in mice

    Energy Technology Data Exchange (ETDEWEB)

    Brugmans, Linda [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Kanaar, Roland [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands); Essers, Jeroen [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands) and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands)]. E-mail: j.essers@erasmusmc.nl

    2007-01-03

    During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.

  7. Efficient and Equitable Exchange in Air Traffic Management Plan Repair using Spender-signed Currency

    NARCIS (Netherlands)

    Jonker, G.M.|info:eu-repo/dai/nl/304832812

    2008-01-01

    Air traffic management (ATM) is concerned with planning of air traffic in the air and on the ground. At any moment up to the moment of execution, a plan may become infeasible, for instance as a result of delays or mechanical failures. In that case a plan needs to be repaired. This may involve delays

  8. Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

    NARCIS (Netherlands)

    Pontier, D.B.

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little

  9. Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

    NARCIS (Netherlands)

    Pontier, D.B.

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little

  10. Homology Requirements and Competition between Gene Conversion and Break-Induced Replication during Double-Strand Break Repair.

    Science.gov (United States)

    Mehta, Anuja; Beach, Annette; Haber, James E

    2017-02-02

    Saccharomyces cerevisiae mating-type switching is initiated by a double-strand break (DSB) at MATa, leaving one cut end perfectly homologous to the HMLα donor, while the second end must be processed to remove a non-homologous tail before completing repair by gene conversion (GC). When homology at the matched end is ≤150 bp, efficient repair depends on the recombination enhancer, which tethers HMLα near the DSB. Thus, homology shorter than an apparent minimum efficient processing segment can be rescued by tethering the donor near the break. When homology at the second end is ≤150 bp, second-end capture becomes inefficient and repair shifts from GC to break-induced replication (BIR). But when pol32 or pif1 mutants block BIR, GC increases 3-fold, indicating that the steps blocked by these mutations are reversible. With short second-end homology, absence of the RecQ helicase Sgs1 promotes gene conversion, whereas deletion of the FANCM-related Mph1 helicase promotes BIR. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery.

    Science.gov (United States)

    Carballo, Jesús A; Panizza, Silvia; Serrentino, Maria Elisabetta; Johnson, Anthony L; Geymonat, Marco; Borde, Valérie; Klein, Franz; Cha, Rita S

    2013-06-01

    An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

  12. Budding yeast ATM/ATR control meiotic double-strand break (DSB levels by down-regulating Rec114, an essential component of the DSB-machinery.

    Directory of Open Access Journals (Sweden)

    Jesús A Carballo

    2013-06-01

    Full Text Available An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs. Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.

  13. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    Science.gov (United States)

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

    Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

  14. Budding yeast ATM/ATR control meiotic double-strand break (DSB) levels by down-regulating Rec114, an essential component of the DSB-machinery.

    OpenAIRE

    Carballo, Jesús A.; Panizza, Silvia; Serrentino, Maria Elisabetta; Anthony L Johnson; Geymonat, Marco; Borde, Valérie; Klein, Franz; Cha, Rita S.

    2013-01-01

    An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, t...

  15. Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

    OpenAIRE

    Johnson, Roger D.; Jasin, Maria

    2000-01-01

    In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contras...

  16. Spontaneous vesicle formation from DSB/AOT mixed system

    Institute of Scientific and Technical Information of China (English)

    CHEN Wenjun; ZHAI Limin; LI Ganzuo; MENG Xiangguang; ZENG Xiancheng

    2003-01-01

    Spontaneous vesicles from the aqueous mixtures of dodecyl sulfonate betaine (DSB) and sodium bis (2-ethylhexyl) sulfosuccinate (AOT) at certain mixingratios have been demonstrated by using calorimetry, freeze-frac- ture transmission electronic microscopy (TEM), negative- staining TEM and dynamic light scattering (DLS) methods. The addition of NaCl will promote vesicle formation and the heat effect of monodisperse vesicle system is greatest. Meanwhile the mechanism was analyzed from the viewpoint of packing parameter f, molecular geometry structure and interaction.

  17. Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

    Directory of Open Access Journals (Sweden)

    Shivani Ponnala

    Full Text Available BACKGROUND: Glioblastoma Multiforme (GBM is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ repair mechanism plays a major role in double strand break (DSB repair in mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU and MMP9-cathepsin B (pMC shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. CONCLUSION/SIGNIFICANCE: Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential

  18. Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

    Science.gov (United States)

    Cesare, Anthony J

    2014-11-01

    Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression.

  19. Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways

    OpenAIRE

    Segatori, Laura; Paukstelis, Paul J.; Gilbert, Hiram F.; Georgiou, George

    2004-01-01

    In the Escherichia coli periplasm, the formation of protein disulfide bonds is catalyzed by DsbA and DsbC. DsbA is a monomer that is maintained in a fully oxidized state by the membrane enzyme DsbB, whereas DsbC is a dimer that is kept reduced by a second membrane protein, DsbD. Although the catalytic regions of DsbA and DsbC are composed of structurally homologous thioredoxin motif domains, DsbA serves only as an oxidase in vivo, whereas DsbC catalyzes disulfide reduction and isomerization a...

  20. Neurons efficiently repair glutamate-induced oxidative DNA damage by a process involving CREB-mediated up-regulation of apurinic endonuclease 1

    DEFF Research Database (Denmark)

    Yang, Jenq-Lin; Tadokoro, Takashi; Keijzers, Guido

    2010-01-01

    damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca(2+)/calmodulin-dependent kinase......-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca(2+)-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors....

  1. The Roles of Several Residues of Escherichia coli DNA Photolyase in the Highly Efficient Photo-Repair of Cyclobutane Pyrimidine Dimers

    Directory of Open Access Journals (Sweden)

    Lei Xu

    2010-01-01

    Full Text Available Escherichia coli DNA photolyase is an enzyme that repairs the major kind of UV-induced lesions, cyclobutane pyrimidine dimer (CPD in DNA utilizing 350–450 nm light as energy source. The enzyme has very high photo-repair efficiency (the quantum yield of the reaction is ~0.85, which is significantly greater than many model compounds that mimic photolyase. This suggests that some residues of the protein play important roles in the photo-repair of CPD. In this paper, we have focused on several residues discussed their roles in catalysis by reviewing the existing literature and some hypotheses.

  2. Preferential repair of DNA double-strand break at the active gene in vivo.

    Science.gov (United States)

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K; Bhaumik, Sukesh R

    2012-10-19

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

  3. CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

    Directory of Open Access Journals (Sweden)

    Hexi Shen

    2017-01-01

    Full Text Available Double-strand breaks (DSBs are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR and nonhomologous end-joining (NHEJ. NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ and the more error-prone KU-independent backup-NHEJ (b-NHEJ pathways, involving the poly (ADP-ribose polymerases (PARPs. However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3 and protoporphyrinogen oxidase (PPO genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80, b-NHEJ (parp1 parp2, or both (ku80 parp1 parp2. We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.

  4. CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants.

    Science.gov (United States)

    Shen, Hexi; Strunks, Gary D; Klemann, Bart J P M; Hooykaas, Paul J J; de Pater, Sylvia

    2017-01-05

    Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants. Copyright © 2017 Shen et al.

  5. Multiple-pathway analysis of double-strand break repair mutations in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dena M Johnson-Schlitz

    2007-04-01

    Full Text Available The analysis of double-strand break (DSB repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage "decision circuit" in which the DSB is first placed into one of two pools from which a specific pathway is then selected.

  6. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

    Science.gov (United States)

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  7. The Vsr endonuclease of Escherichia coli: an efficient DNA repair enzyme and a potent mutagen.

    Science.gov (United States)

    Macintyre, G; Doiron, K M; Cupples, C G

    1997-01-01

    The Vsr endonuclease of Escherichia coli initiates the repair of T/G mismatches caused by deamination of 5-methylcytosine to thymine. In this paper, we examine the capacity of Vsr to prevent CG-to-TA mutations in cells with increased transcription of the cytosine methylase gene (dcm). We find that sufficient Vsr is produced by a single chromosomal copy of vsr to prevent mutagenesis. We also investigate the cause of the transition and frameshift mutations in cells overproducing Vsr. Neither the absence of the dcm methylase nor its overproduction affects Vsr-stimulated mutagenesis. However, addition of mutS, mutL, or mutH on multicopy plasmids has a significant effect: mutL or mutH decreases the number of mutations, while mutS stimulates mutagenesis. The mut-containing plasmids have the same effect in cells treated with 2-aminopurine and in cells made defective in DNA proofreading, two experimental situations known to cause transition and frameshift mutations by saturating mismatch repair. PMID:9324251

  8. A miR-590/Acvr2a/Rad51b Axis Regulates DNA Damage Repair during mESC Proliferation

    Directory of Open Access Journals (Sweden)

    Qidong Liu

    2014-12-01

    Full Text Available Embryonic stem cells (ESCs enable rapid proliferation that also causes DNA damage. To maintain genomic stabilization during rapid proliferation, ESCs must have an efficient system to repress genotoxic stress. Here, we show that withdrawal of leukemia inhibitory factor (LIF, which maintains the self-renewal capability of mouse ESCs (mESCs, significantly inhibits the cell proliferation and DNA damage of mESCs and upregulates the expression of miR-590. miR-590 promotes single-strand break (SSB and double-strand break (DSB damage repair, thus slowing proliferation of mESCs without influencing stemness. miR-590 directly targets Activin receptor type 2a (Acvr2a to mediate Activin signaling. We identified the homologous recombination-mediated repair (HRR gene, Rad51b, as a downstream molecule of the miR-590/Acvr2a pathway regulating the SSB and DSB damage repair and cell cycle. Our study shows that a miR-590/Acvr2a/Rad51b signaling axis ensures the stabilization of mESCs by balancing DNA damage repair and rapid proliferation during self-renewal.

  9. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes.

    Science.gov (United States)

    Ono, Ryuichi; Ishii, Masayuki; Fujihara, Yoshitaka; Kitazawa, Moe; Usami, Takako; Kaneko-Ishino, Tomoko; Kanno, Jun; Ikawa, Masahito; Ishino, Fumitoshi

    2015-07-28

    The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

  10. Correlation between slowly repairable double-strand breaks and thermal radiosensitization in the human HeLa S3 cell line

    NARCIS (Netherlands)

    Kampinga, HH; Hiemstra, YS; Konings, AWT; Dikomey, E

    1997-01-01

    The effect of heat on double-strand breaks (dsb) repair was compared with thermal radiosensitization using HeLa S3 cells. Cells were exposed to a combined treatment of X-irradiation followed by heat (44 degrees C, 0.5 h) separated by time intervals up to 8h. DNA dsb were measured by PFGE and surviva

  11. Evaluation of the efficiency of a repaired chloride-contaminated concrete

    Directory of Open Access Journals (Sweden)

    Andrade, C.

    1988-09-01

    Full Text Available This report describes several tests that are being carried out on a viaduct near Madrid City, whose reinforcements have been seriously damaged by corrosion. These tests consist in measuring corrosion potentials and in a time soon to come, they will include the measurement of corrosion rates by means of the apparent Polarization Resistance. A methodology of laboratory tests to evaluate the protective capacity of the repairing system used towards the reinforcements is also included. Tests results have reported that the repairing materials employed do not fit the repassivating and protective function they should satisfy. Finally, it is also recalled to attention the real need in developing suitable test specifications able to provide a characterization of these repairing materials when they are going to be employed in structures damaged by corrosion in reinforcements.

    En el presente trabajo se describen los ensayos que se están efectuando en un viaducto de las cercanías de Madrid y que tía sufrido importantes daños por corrosión de armaduras. Estos Ensayos consisten en la medida de los potenciales de corrosión y en un próximo futuro, de la velocidad de corrosión (método de la Resistencia de Polarización Aparente. También se presenta la metodología de los ensayos de laboratorio efectuados para caracterizar la capacidad protectora hacia las armaduras del sistema de reparación utilizado en el viaducto. Los resultado de estos ensayos han mostrado que los materiales de reparación no resultan idóneos para la función repasivante y protectora que deberán desempeñar. Se destaca, finalmente, la necesidad de desarrollar una normativa de ensayos adecuada que permita caracterizar a estos materiales de reparación cuando van a ser usados en obras dañadas por corrosión de armaduras.

  12. Nonhomologous end-joining repair plays a more important role than homologous recombination repair in defining radiosensitivity after exposure to high-LET radiation.

    Science.gov (United States)

    Takahashi, Akihisa; Kubo, Makoto; Ma, Hongyu; Nakagawa, Akiko; Yoshida, Yukari; Isono, Mayu; Kanai, Tatsuaki; Ohno, Tatsuya; Furusawa, Yoshiya; Funayama, Tomoo; Kobayashi, Yasuhiko; Nakano, Takashi

    2014-09-01

    DNA double-strand breaks (DSBs) induced by ionizing radiation pose a major threat to cell survival. The cell can respond to the presence of DSBs through two major repair pathways: homologous recombination (HR) and nonhomologous end joining (NHEJ). Higher levels of cell death are induced by high-linear energy transfer (LET) radiation when compared to low-LET radiation, even at the same physical doses, due to less effective and efficient DNA repair. To clarify whether high-LET radiation inhibits all repair pathways or specifically one repair pathway, studies were designed to examine the effects of radiation with different LET values on DNA DSB repair and radiosensitivity. Embryonic fibroblasts bearing repair gene (NHEJ-related Lig4 and/or HR-related Rad54) knockouts (KO) were used and their responses were compared to wild-type cells. The cells were exposed to X rays, spread-out Bragg peak (SOBP) carbon ion beams as well as with carbon, iron, neon and argon ions. Cell survival was measured with colony-forming assays. The sensitization enhancement ratio (SER) values were calculated using the 10% survival dose of wild-type cells and repair-deficient cells. Cellular radiosensitivity was listed in descending order: double-KO cells > Lig4-KO cells > Rad54-KO cells > wild-type cells. Although Rad54-KO cells had an almost constant SER value, Lig4-KO cells showed a high-SER value when compared to Rad54-KO cells, even with increasing LET values. These results suggest that with carbon-ion therapy, targeting NHEJ repair yields higher radiosensitivity than targeting homologous recombination repair.

  13. Mismatch repair in Streptococcus pneumoniae: relationship between base mismatches and transformation efficiencies.

    Science.gov (United States)

    Claverys, J P; Méjean, V; Gasc, A M; Sicard, A M

    1983-10-01

    Genetic transformation in Streptococcus pneumoniae involves the insertion of single-stranded pieces of donor DNA into a recipient genome. Efficiencies of transformation strongly depend on the mutations (markers) carried by donor DNA. Markers are classified according to their transforming efficiencies into very high, high, intermediate, and low efficiency. The last is approximately 1/20th as efficient as the first. This marker effect is under the control of the Hex system, which is thought to correct mismatches at the donor-recipient heteroduplex stage in transformation. To investigate this effect, wild type, mutant, and revertant DNA sequences at five genetic sites within the amiA locus were determined. The results show that low-efficiency markers arise from transitional changes A . T to G . C. The transversion A . T to T . A corresponds to an intermediate-efficiency marker. Transversions G . C to T . A and G . C to C . G lead to high-efficiency markers. Among the eight possible mismatches that could exist transiently at the heteroduplex stage in transformation, only two--namely, A/G and C/C--are not corrected by the Hex system. It is noteworthy that the four possible base pairs (A . T, T . A, G . C, and C . G) have been encountered at the very same site (amiA6 site), which constitutes a good illustration of the marker effect. DNA sequence analysis also reveals that short deletions (33 or 34 bases long) are integrated with very high efficiencies. These results confirm that the Hex system corrects point mismatches harbored in donor-recipient heteroduplexes thousands of bases long. The correction pattern of the Hex system toward multiple-base mismatches has also been investigated. Its behavior toward double-base mismatches is complex, suggesting that neighboring sequences may affect the detection of mispaired bases.

  14. Application of k-person and k-task maximal efficiency assignment algorithm to water piping repair

    Directory of Open Access Journals (Sweden)

    Su-juan ZHENG

    2009-06-01

    Full Text Available Solving the absent assignment problem of the shortest time limit in a weighted bipartite graph with the minimal weighted k-matching algorithm is unsuitable for situations in which large numbers of problems need to be addressed by large numbers of parties. This paper simplifies the algorithm of searching for the even alternating path that contains a maximal element using the minimal weighted k-matching theorem and intercept graph. A program for solving the maximal efficiency assignment problem was compiled. As a case study, the program was used to solve the assignment problem of water piping repair in the case of a large number of companies and broken pipes, and the validity of the program was verified.

  15. Application of k-person and k-task maximal efficiency assignment algorithm to water piping repair

    Institute of Scientific and Technical Information of China (English)

    Su-juan ZHENG; Xiu-ming YU; Li-qing CAO

    2009-01-01

    Solving the absent assignment problern of the shortest time limit in a weighted bipartite graph with the minimal weighted k-matching algorithm is unsuitable for situations in which large numbers of problems need to be addressed by large numbers of parties. This paper simplifies the algorithm of searching for the even alternating path that contains a maximal element using the minimal weighted k-matching theorem and intercept graph. A program for solving the maximal efficiency assignment problem was compiled. As a case study, the program was used to solve the assignment problem of water piping repair in the case of a large number of companies and broken pipes, and the validity of the program was verified.

  16. Control of gene editing by manipulation of DNA repair mechanisms.

    Science.gov (United States)

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  17. Negative Regulation of DsbA-L Gene Expression by the Transcription Factor Sp1

    OpenAIRE

    Fang, Qichen; Yang, Wenjing; Li, Huating; Hu, Wenxiu; Chen, Lihui; Jiang, Shan; Dong, Kun; Song, Qianqian; Wang, Chen; Chen, Shuo; LIU, FENG; Jia, Weiping

    2014-01-01

    Disulfide-bond A oxidoreductase-like protein (DsbA-L) possesses beneficial effects such as promoting adiponectin multimerization and stability, increasing insulin sensitivity, and enhancing energy metabolism. The expression level of DsbA-L is negatively correlated with obesity in mice and humans, but the underlying mechanisms remain unknown. To address this question, we generated reporter gene constructs containing the promoter sequence of the mouse DsbA-L gene. Deletion analysis showed that ...

  18. Nampt is involved in DNA double-strand break repair

    Institute of Scientific and Technical Information of China (English)

    Bingtao Zhu; Xiaoli Deng; Yifan Sun; Lin Bai; Zhikai Xiahou; Yusheng Cong; Xingzhi Xu

    2012-01-01

    DNA double-strand break (DSB) is the most severe form of DNA damage,which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ).Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer.Nicotinamide phosphoribosyltransferase (Nampt),which is involved in nicotinamide adenine dinucleotide metabolism,is overexpressed in a variety of tumors.In this report,we found that Nampt physically associated with CtlP and DNA-PKcs/Ku80,which are key factors in HR and NHEJ,respectively.Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair.Furthermore,the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining,indicating a delay in the onset of cellular senescence in normal human fibroblasts.Taken together,our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair,contributing to the acceleration of cellular senescence.

  19. Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells.

    Science.gov (United States)

    Wang, Yebo; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

    2016-11-28

    Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.

  20. Characterization of new DsbB-like thiol-oxidoreductases of Campylobacter jejuni and Helicobacter pylori and classification of the DsbB family based on phylogenomic, structural and functional criteria.

    Science.gov (United States)

    Raczko, Anna M; Bujnicki, Janusz M; Pawlowski, Marcin; Godlewska, Renata; Lewandowska, Magdalena; Jagusztyn-Krynicka, Elzbieta K

    2005-01-01

    In Gram-negative bacterial cells, disulfide bond formation occurs in the oxidative environment of the periplasm and is catalysed by Dsb (disulfide bond) proteins found in the periplasm and in the inner membrane. In this report the identification of a new subfamily of disulfide oxidoreductases encoded by a gene denoted dsbI, and functional characterization of DsbI proteins from Campylobacter jejuni and Helicobacter pylori, as well as DsbB from C. jejuni, are described. The N-terminal domain of DsbI is related to DsbB proteins and comprises five predicted transmembrane segments, while the C-terminal domain is predicted to locate to the periplasm and to fold into a beta-propeller structure. The dsbI gene is co-transcribed with a small ORF designated dba (dsbI-accessory). Based on a series of deletion and complementation experiments it is proposed that DsbB can complement the lack of DsbI but not the converse. In the presence of DsbB, the activity of DsbI was undetectable, hence it probably acts only on a subset of possible substrates of DsbB. To reconstruct the principal events in the evolution of DsbB and DsbI proteins, sequences of all their homologues identifiable in databases were analysed. In the course of this study, previously undetected variations on the common thiol-oxidoreductase theme were identified, such as development of an additional transmembrane helix and loss or migration of the second pair of Cys residues between two distinct periplasmic loops. In conjunction with the experimental characterization of two members of the DsbI lineage, this analysis has resulted in the first comprehensive classification of the DsbB/DsbI family based on structural, functional and evolutionary criteria.

  1. Rapid MCNP simulation of DNA double strand break (DSB) relative biological effectiveness (RBE) for photons, neutrons, and light ions.

    Science.gov (United States)

    Stewart, Robert D; Streitmatter, Seth W; Argento, David C; Kirkby, Charles; Goorley, John T; Moffitt, Greg; Jevremovic, Tatjana; Sandison, George A

    2015-11-07

    To account for particle interactions in the extracellular (physical) environment, information from the cell-level Monte Carlo damage simulation (MCDS) for DNA double strand break (DSB) induction has been integrated into the general purpose Monte Carlo N-particle (MCNP) radiation transport code system. The effort to integrate these models is motivated by the need for a computationally efficient model to accurately predict particle relative biological effectiveness (RBE) in cell cultures and in vivo. To illustrate the approach and highlight the impact of the larger scale physical environment (e.g. establishing charged particle equilibrium), we examined the RBE for DSB induction (RBEDSB) of x-rays, (137)Cs γ-rays, neutrons and light ions relative to γ-rays from (60)Co in monolayer cell cultures at various depths in water. Under normoxic conditions, we found that (137)Cs γ-rays are about 1.7% more effective at creating DSB than γ-rays from (60)Co (RBEDSB  =  1.017) whereas 60-250 kV x-rays are 1.1 to 1.25 times more efficient at creating DSB than (60)Co. Under anoxic conditions, kV x-rays may have an RBEDSB up to 1.51 times as large as (60)Co γ-rays. Fission neutrons passing through monolayer cell cultures have an RBEDSB that ranges from 2.6 to 3.0 in normoxic cells, but may be as large as 9.93 for anoxic cells. For proton pencil beams, Monte Carlo simulations suggest an RBEDSB of about 1.2 at the tip of the Bragg peak and up to 1.6 a few mm beyond the Bragg peak. Bragg peak RBEDSB increases with decreasing oxygen concentration, which may create opportunities to apply proton dose painting to help address tumor hypoxia. Modeling of the particle RBE for DSB induction across multiple physical and biological scales has the potential to aid in the interpretation of laboratory experiments and provide useful information to advance the safety and effectiveness of hadron therapy in the treatment of cancer.

  2. Rapid MCNP simulation of DNA double strand break (DSB) relative biological effectiveness (RBE) for photons, neutrons, and light ions

    Science.gov (United States)

    Stewart, Robert D.; Streitmatter, Seth W.; Argento, David C.; Kirkby, Charles; Goorley, John T.; Moffitt, Greg; Jevremovic, Tatjana; Sandison, George A.

    2015-11-01

    To account for particle interactions in the extracellular (physical) environment, information from the cell-level Monte Carlo damage simulation (MCDS) for DNA double strand break (DSB) induction has been integrated into the general purpose Monte Carlo N-particle (MCNP) radiation transport code system. The effort to integrate these models is motivated by the need for a computationally efficient model to accurately predict particle relative biological effectiveness (RBE) in cell cultures and in vivo. To illustrate the approach and highlight the impact of the larger scale physical environment (e.g. establishing charged particle equilibrium), we examined the RBE for DSB induction (RBEDSB) of x-rays, 137Cs γ-rays, neutrons and light ions relative to γ-rays from 60Co in monolayer cell cultures at various depths in water. Under normoxic conditions, we found that 137Cs γ-rays are about 1.7% more effective at creating DSB than γ-rays from 60Co (RBEDSB  =  1.017) whereas 60-250 kV x-rays are 1.1 to 1.25 times more efficient at creating DSB than 60Co. Under anoxic conditions, kV x-rays may have an RBEDSB up to 1.51 times as large as 60Co γ-rays. Fission neutrons passing through monolayer cell cultures have an RBEDSB that ranges from 2.6 to 3.0 in normoxic cells, but may be as large as 9.93 for anoxic cells. For proton pencil beams, Monte Carlo simulations suggest an RBEDSB of about 1.2 at the tip of the Bragg peak and up to 1.6 a few mm beyond the Bragg peak. Bragg peak RBEDSB increases with decreasing oxygen concentration, which may create opportunities to apply proton dose painting to help address tumor hypoxia. Modeling of the particle RBE for DSB induction across multiple physical and biological scales has the potential to aid in the interpretation of laboratory experiments and provide useful information to advance the safety and effectiveness of hadron therapy in the treatment of cancer.

  3. Inter-individual variation in DNA double-strand break repair in human fibroblasts before and after exposure to low doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Paul F.; Nham, Peter B.; Urbin, Salustra S.; Hinz, John M.; Jones, Irene M. [Biosciences and Biotechnology Division, PO Box 808, L-452, Lawrence Livermore National Laboratory, Livermore, CA, 94551-0808 (United States); Thompson, Larry H., E-mail: thompson14@llnl.gov [Biosciences and Biotechnology Division, PO Box 808, L-452, Lawrence Livermore National Laboratory, Livermore, CA, 94551-0808 (United States)

    2010-01-05

    DNA double-strand breaks (DSB) are generally considered the most critical lesion induced by ionizing radiation (IR) and may initiate carcinogenesis and other disease. Using an immunofluorescence assay to simultaneously detect nuclear foci of the phosphorylated forms of histone H2AX and ATM kinase at sites of DSBs, we examined the response of 25 apparently normal and 10 DNA repair-deficient (ATM, ATR, NBN, LIG1, LIG4, and FANCG) primary fibroblast strains irradiated with low doses of {sup 137}Cs {gamma}-rays. Quiescent G{sub 0}/G{sub 1}-phase cultures were exposed to 5, 10, and 25 cGy and allowed to repair for 24 h. The maximum level of IR-induced foci (0.15 foci per cGy, at 10 or 30 min) in the normal strains showed much less inter-individual variation (CV {approx} 0.2) than the level of spontaneous foci, which ranged from 0.2-2.6 foci/cell (CV {approx} 0.6; mean {+-} SD of 1.00 {+-} 0.57). Significantly slower focus formation post-irradiation was observed in seven normal strains, similar to most mutant strains examined. There was variation in repair efficiency measured by the fraction of IR-induced foci remaining 24 h post-irradiation, curiously with the strains having slower focus formation showing more efficient repair after 25 cGy. Interestingly, the ranges of spontaneous and residual induced foci levels at 24 h in the normal strains were as least as large as those observed for the repair-defective mutant strains. The inter-individual variation in DSB foci parameters observed in cells exposed to low doses of ionizing radiation in this small survey of apparently normal people suggests that hypomorphic genetic variants in genomic maintenance and/or DNA damage signaling and repair genes may contribute to differential susceptibility to cancer induced by environmental mutagens.

  4. Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint.

    Directory of Open Access Journals (Sweden)

    Ericca L Stamper

    Full Text Available Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs. DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.

  5. Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint.

    Science.gov (United States)

    Stamper, Ericca L; Rodenbusch, Stacia E; Rosu, Simona; Ahringer, Julie; Villeneuve, Anne M; Dernburg, Abby F

    2013-01-01

    Meiotic recombination, an essential aspect of sexual reproduction, is initiated by programmed DNA double-strand breaks (DSBs). DSBs are catalyzed by the widely-conserved Spo11 enzyme; however, the activity of Spo11 is regulated by additional factors that are poorly conserved through evolution. To expand our understanding of meiotic regulation, we have characterized a novel gene, dsb-1, that is specifically required for meiotic DSB formation in the nematode Caenorhabditis elegans. DSB-1 localizes to chromosomes during early meiotic prophase, coincident with the timing of DSB formation. DSB-1 also promotes normal protein levels and chromosome localization of DSB-2, a paralogous protein that plays a related role in initiating recombination. Mutations that disrupt crossover formation result in prolonged DSB-1 association with chromosomes, suggesting that nuclei may remain in a DSB-permissive state. Extended DSB-1 localization is seen even in mutants with defects in early recombination steps, including spo-11, suggesting that the absence of crossover precursors triggers the extension. Strikingly, failure to form a crossover precursor on a single chromosome pair is sufficient to extend the localization of DSB-1 on all chromosomes in the same nucleus. Based on these observations we propose a model for crossover assurance that acts through DSB-1 to maintain a DSB-permissive state until all chromosome pairs acquire crossover precursors. This work identifies a novel component of the DSB machinery in C. elegans, and sheds light on an important pathway that regulates DSB formation for crossover assurance.

  6. Delayed repair of radiation induced clustered DNA damage: Friend or foe?

    Energy Technology Data Exchange (ETDEWEB)

    Eccles, Laura J., E-mail: laura.eccles@rob.ox.ac.uk [DNA Damage Group, Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom); O' Neill, Peter, E-mail: peter.oneill@rob.ox.ac.uk [DNA Damage Group, Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom); Lomax, Martine E., E-mail: martine.lomax@rob.ox.ac.uk [DNA Damage Group, Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ (United Kingdom)

    2011-06-03

    A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with associated base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will concentrate on the experimental findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addition certain non-DSB clustered damaged sites are processed within the cell to form additional DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a 'friend', leading to cell killing in tumour cells or as a 'foe', resulting in the formation of mutations and genetic instability in normal tissue.

  7. Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander;

    2013-01-01

    single-strand breaks (SSBs) and double-strand breaks (DSBs) in human peripheral blood mononuclear cells (PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ-H2AX response, and DSB repair capacity...... in a study population consisting of 216 individuals from a population-based sample of twins aged 40-77 years. Age in this range did not seem to have any effect on the SSB parameters. However, γ-H2AX response and DSB repair capacity decreased with increasing age, although the associations did not reach...

  8. DNA double strand break repair, chromosome synapsis and transcriptional silencing in meiosis.

    Science.gov (United States)

    Inagaki, Akiko; Schoenmakers, Sam; Baarends, Willy M

    2010-05-16

    Chromosome pairing and synapsis during meiotic prophase requires the formation and repair of DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme SPO11. Chromosomes, or chromosomal regions, that lack a pairing partner, such as the largely heterologous X and Y chromosomes, show delayed meiotic DSB repair and are transcriptionally silenced. Herein, we review meiosis-specific aspects of DSB repair in relation to homology recognition and meiotic silencing of heterologous regions. We propose a dynamic interplay between progression of synapsis and persistent meiotic DSBs. Signaling from these persistent breaks could inhibit heterologous synapsis and stimulate meiotic silencing of the X and Y chromosomes.

  9. Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair

    Science.gov (United States)

    Zhu, Kai; Lai, Hao; Guo, Changfa; Li, Jun; Wang, Yulin; Wang, Lingyan; Wang, Chunsheng

    2014-01-01

    Mesenchymal stem cell (MSC) transplantation has attracted much attention in myocardial infarction therapy. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. Small interfering RNA-mediated prolyl hydroxylase domain protein 2 (PHD2) silencing in MSCs holds tremendous potential to enhance their survival and paracrine effect after transplantation. However, an efficient and biocompatible PHD2 silencing system for clinical application is lacking. Herein, we developed a novel PHD2 silencing system based on arginine-terminated generation 4 poly(amidoamine) (Arg-G4) nanoparticles. The system exhibited effective and biocompatible small interfering RNA delivery and PHD2 silencing in MSCs in vitro. After genetically modified MSC transplantation in myocardial infarction models, MSC survival and paracrine function of IGF-1 were enhanced significantly in vivo. As a result, we observed decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angiogenesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This work demonstrated that an Arg-G4 nanovector-based PHD2 silencing system could enhance the efficiency of MSC transplantation for infarcted myocardium repair. PMID:25429216

  10. Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange

    NARCIS (Netherlands)

    H.B. Beverloo (Berna); R.D. Johnson (Roger); M. Jasin (Maria); R. Kanaar (Roland); J.H.J. Hoeijmakers (Jan); M.L.G. Dronkert (Mies)

    2000-01-01

    textabstractCells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we

  11. Cell resistance to the Cytolethal Distending Toxin involves an association of DNA repair mechanisms

    Science.gov (United States)

    Bezine, Elisabeth; Malaisé, Yann; Loeuillet, Aurore; Chevalier, Marianne; Boutet-Robinet, Elisa; Salles, Bernard; Mirey, Gladys; Vignard, Julien

    2016-01-01

    The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways. Here, we validate the involvement of the two major DSB repair mechanisms, Homologous Recombination and Non Homologous End Joining, in the management of CDT-induced lesions. We show that impairment of single-strand break repair (SSBR), but not nucleotide excision repair, sensitizes cells to CDT, and we explore the interplay of SSBR with the DSB repair mechanisms. Finally, we document the role of the replicative stress response and demonstrate the involvement of the Fanconi Anemia repair pathway in response to CDT. In conclusion, our work indicates that cellular survival to CDT-induced DNA damage involves different repair pathways, in particular SSBR. This reinforces a model where CDT-related genotoxicity primarily involves SSBs rather than DSBs, underlining the importance of cell proliferation during CDT intoxication and pathogenicity. PMID:27775089

  12. 53BP1 fosters fidelity of homology-directed DNA repair

    DEFF Research Database (Denmark)

    Ochs, Fena; Somyajit, Kumar; Altmeyer, Matthias

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair...

  13. ERCC1-XPF endonuclease facilitates DNA double-strand break repair.

    Science.gov (United States)

    Ahmad, Anwaar; Robinson, Andria Rasile; Duensing, Anette; van Drunen, Ellen; Beverloo, H Berna; Weisberg, David B; Hasty, Paul; Hoeijmakers, Jan H J; Niedernhofer, Laura J

    2008-08-01

    ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and gammaH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1(-/-) Ku86(-/-) fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3' overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.

  14. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography.

    Science.gov (United States)

    Thompson, Larry H

    2012-01-01

    The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.

  15. A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair.

    Directory of Open Access Journals (Sweden)

    Michèle Telorack

    2016-01-01

    Full Text Available The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing.

  16. Tid1/Rdh54 translocase is phosphorylated through a Mec1- and Rad53-dependent manner in the presence of DSB lesions in budding yeast.

    Science.gov (United States)

    Ferrari, Matteo; Nachimuthu, Benjamin Tamilselvan; Donnianni, Roberto Antonio; Klein, Hannah; Pellicioli, Achille

    2013-05-01

    Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Tid1/Rdh54, a member of the Swi2-like family proteins, has an ATPase activity and may contribute to the remodelling of nucleosomes on DNA. Tid1 dislocates Rad51 recombinase from dsDNA, can unwind and supercoil DNA filaments, and has been implicated in checkpoint adaptation from a G2/M arrest induced by an unrepaired DSB. Here we show that both ATR/Mec1 and Chk2/Rad53 kinases are implicated in the phosphorylation of Tid1 in the presence of DNA damage, indicating that the protein is regulated during the DNA damage response. We show that Tid1 ATPase activity is dispensable for its phosphorylation and for its recruitment near a DSB, but it is required to switch off Rad53 activation and for checkpoint adaptation. Mec1 and Rad53 kinases, together with Rad51 recombinase, are also implicated in the hyper-phosphorylation of the ATPase defective Tid1-K318R variant and in the efficient binding of the protein to the DSB site. In summary, Tid1 is a novel target of the DNA damage checkpoint pathway that is also involved in checkpoint adaptation.

  17. DNA repair: Dynamic defenders against cancer and aging

    Energy Technology Data Exchange (ETDEWEB)

    Fuss, Jill O.; Cooper, Priscilla K.

    2006-04-01

    (UV) component of sunlight. NER can be divided into two classes based on where the repair occurs. NER occurring in DNA that is not undergoing transcription (i.e., most of the genome) is called global genome repair (GGR or GGNER), while NER taking place in the transcribed strand of active genes is called transcription-coupled repair (TCR or TC-NER). We will explore NER in more detail below. Mismatch repair (MMR) is another type of excision repair that specifically removes mispaired bases resulting from replication errors. DNA damage can also result in breaks in the DNA backbone, in one or both strands. Single-strand breaks (SSBs) are efficiently repaired by a mechanism that shares common features with the later steps in BER. Double-strand breaks (DSBs) are especially devastating since by definition there is no intact complementary strand to serve as a template for repair, and even one unrepaired DSB can be lethal [3]. In cells that have replicated their DNA prior to cell division, the missing information can be supplied by the duplicate copy, or sister chromatid, and DSBs in these cells are faithfully repaired by homologous recombination involving the exchange of strands of DNA between the two copies. However, most cells in the body are non-dividing, and in these cells the major mechanism for repairing DSBs is by non-homologous end joining (NHEJ), which as the name implies involves joining two broken DNA ends together without a requirement for homologous sequence and which therefore has a high potential for loss of genetic information.

  18. An estimate of maintenance efficiency in Brown-Proschan imperfect repair model with bathtub failure intensity

    Directory of Open Access Journals (Sweden)

    Makram Krit

    2012-06-01

    Full Text Available Purpose: Estimate the maintenance efficiency in the Brown-Proschan model with the bathtub failure intensity.Design/methodology/approach: Empirical research through which we propose a framework to establish the characteristics of failure process and its influence on maintenance process.Findings: The main contribution of the present study is the reformulation of the Brown and Proschan model using the bathtub failure intensityPractical implications: Our model is defined by BP reformulation one using bathtub failure intensity. This form of intensity is presented like superposition of two NHPP and Homogeneous Poisson one.Originality/value: This is the follow on research on the study that employed the power-law-process type of failure intensity.

  19. Human INO80 chromatin-remodelling complex contributes to DNA double-strand break repair via the expression of Rad54B and XRCC3 genes.

    Science.gov (United States)

    Park, Eun-Jung; Hur, Shin-Kyoung; Kwon, Jongbum

    2010-10-15

    Recent studies have shown that the SWI/SNF family of ATP-dependent chromatin-remodelling complexes play important roles in DNA repair as well as in transcription. The INO80 complex, the most recently described member of this family, has been shown in yeast to play direct role in DNA DSB (double-strand break) repair without affecting the expression of the genes involved in this process. However, whether this function of the INO80 complex is conserved in higher eukaryotes has not been investigated. In the present study, we found that knockdown of hINO80 (human INO80) confers DNA-damage hypersensitivity and inefficient DSB repair. Microarray analysis and other experiments have identified the Rad54B and XRCC3 (X-ray repair complementing defective repair in Chinese-hamster cells 3) genes, implicated in DSB repair, to be repressed by hINO80 deficiency. Chromatin immunoprecipitation studies have shown that hINO80 binds to the promoters of the Rad54B and XRCC3 genes. Re-expression of the Rad54B and XRCC3 genes rescues the DSB repair defect in hINO80-deficient cells. These results suggest that hINO80 assists DSB repair by positively regulating the expression of the Rad54B and XRCC3 genes. Therefore, unlike yeast INO80, hINO80 can contribute to DSB repair indirectly via gene expression, suggesting that the mechanistic role of this chromatin remodeller in DSB repair is evolutionarily diversified.

  20. Selective targeting of homologous DNA recombination repair by gemcitabine

    NARCIS (Netherlands)

    Wachters, FM; van Putten, JWG; Maring, JG; Zdzienicka, MZ; Groen, HJM; Kampinga, HH

    2003-01-01

    Purpose: Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a potent radiosensitizer. The mechanism of dFdC-mediated radiosensitization is yet poorly understood. We recently excluded inhibition of DNA double-strand break (DSB) repair by nonhomologous end-joining (NHEJ) as a means of

  1. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

    Science.gov (United States)

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R.; Guerrero, Justin A.; Shen, Tian; Casali, Paolo

    2017-01-01

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52−/− B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S–S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S–S recombination, as emphasized by the significantly greater CSR reduction in Rad52−/− versus Rad52+/+ B cells on Ku86 knockdown. PMID:28176781

  2. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination.

    Science.gov (United States)

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R; Guerrero, Justin A; Shen, Tian; Casali, Paolo

    2017-02-08

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S-S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52(+/+) counterparts, which display normal CSR, Rad52(-/-) B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S-S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S-S recombination, as emphasized by the significantly greater CSR reduction in Rad52(-/-) versus Rad52(+/+) B cells on Ku86 knockdown.

  3. Comparing Benign and Malignant Neoplasia and DSB Induction for Low-and High-LET Radiation

    Science.gov (United States)

    Burns, Fredric; (Eric) Tang, Moon-Shong; Wu, Feng

    One-and 2-stage models based on DNA double strand breaks (DSBs) have been developed to describe the dose and LET dependence of cancer induction in rat skin exposed to the Bragg plateau of several ion beams or electron radiation. Data are presented showing that carcinomas (malignant) and fibromas (benign) are induced differently by low and high LET radiation. DSBs are subject to complex repair processes, including homologous and non-homologous end joining, that slowly eliminate broken chromosome ends but at the expense of elevating genomic instability that increases the risk of neoplasia. In this formulation the initial molecular lesion in radiation carcinogenesis is assumed to be a DNA double strand break (DSB). The 2-event model assumes that pairs of DSBs join to create cellular genomic instability that eventually progresses to malignancy. The 1-event model assumes that joining is insignificant but that unrepaired DSBs remain and are sufficiently destabilizing to produce low-grade neoplasias. The respective expected relationships between neoplasia yield (Y), radiation dose (D) and LET (L) are: Y(D) = CLD + BD2 (A) for 2-events and Y(D) = CLD (B) for 1-event. Respective B and C values have been evaluated empirically for carcinomas, fibromas and DSBs, the latter via the -H2Ax technique in surrogate keratinocytes, for several types of radiations, including, 40Ar ions, 56Fe ions, 20Ne ions, protons, electrons and x-rays. Fibromas outnumber carcinomas by about 6:1 but are more sensitive than carcinomas to the cytolethal effect of the radiations. The 2-event model agrees well with carcinoma yields in rat skin but fails to model fibromas correctly. Instead the fibroma yields best fitted with the 1-event model for the high LET ion radiations, but at very low LET (electron radiation), an empirical D3 component becomes apparent which is not currently incorporated into the theoretical model. At higher LET values, the D3 component was not detected. The overall results are

  4. Modeling damage complexity-dependent non-homologous end-joining repair pathway.

    Directory of Open Access Journals (Sweden)

    Yongfeng Li

    Full Text Available Non-homologous end joining (NHEJ is the dominant DNA double strand break (DSB repair pathway and involves several repair proteins such as Ku, DNA-PKcs, and XRCC4. It has been experimentally shown that the choice of NHEJ proteins is determined by the complexity of DSB. In this paper, we built a mathematical model, based on published data, to study how NHEJ depends on the damage complexity. Under an appropriate set of parameters obtained by minimization technique, we can simulate the kinetics of foci track formation in fluorescently tagged mammalian cells, Ku80-EGFP and DNA-PKcs-YFP for simple and complex DSB repair, respectively, in good agreement with the published experimental data, supporting the notion that simple DSB undergo fast repair in a Ku-dependent, DNA-PKcs-independent manner, while complex DSB repair requires additional DNA-PKcs for end processing, resulting in its slow repair, additionally resulting in slower release rate of Ku and the joining rate of complex DNA ends. Based on the numerous experimental descriptions, we investigated several models to describe the kinetics for complex DSB repair. An important prediction of our model is that the rejoining of complex DSBs is through a process of synapsis formation, similar to a second order reaction between ends, rather than first order break filling/joining. The synapsis formation (SF model allows for diffusion of ends before the synapsis formation, which is precluded in the first order model by the rapid coupling of ends. Therefore, the SF model also predicts the higher number of chromosomal aberrations observed with high linear energy transfer (LET radiation due to the higher proportion of complex DSBs compared to low LET radiation, and an increased probability of misrejoin following diffusion before the synapsis is formed, while the first order model does not provide a mechanism for the increased effectiveness in chromosomal aberrations observed.

  5. Patching Broken DNA: Nucleosome Dynamics and the Repair of DNA Breaks.

    Science.gov (United States)

    Gursoy-Yuzugullu, Ozge; House, Nealia; Price, Brendan D

    2016-05-08

    The ability of cells to detect and repair DNA double-strand breaks (DSBs) is dependent on reorganization of the surrounding chromatin structure by chromatin remodeling complexes. These complexes promote access to the site of DNA damage, facilitate processing of the damaged DNA and, importantly, are essential to repackage the repaired DNA. Here, we will review the chromatin remodeling steps that occur immediately after DSB production and that prepare the damaged chromatin template for processing by the DSB repair machinery. DSBs promote rapid accumulation of repressive complexes, including HP1, the NuRD complex, H2A.Z and histone methyltransferases at the DSB. This shift to a repressive chromatin organization may be important to inhibit local transcription and limit mobility of the break and to maintain the DNA ends in close contact. Subsequently, the repressive chromatin is rapidly dismantled through a mechanism involving dynamic exchange of the histone variant H2A.Z. H2A.Z removal at DSBs alters the acidic patch on the nucleosome surface, promoting acetylation of the H4 tail (by the NuA4-Tip60 complex) and shifting the chromatin to a more open structure. Further, H2A.Z removal promotes chromatin ubiquitination and recruitment of additional DSB repair proteins to the break. Modulation of the nucleosome surface and nucleosome function during DSB repair therefore plays a vital role in processing of DNA breaks. Further, the nucleosome surface may function as a central hub during DSB repair, directing specific patterns of histone modification, recruiting DNA repair proteins and modulating chromatin packing during processing of the damaged DNA template.

  6. Nanovector-based prolyl hydroxylase domain 2 silencing system enhances the efficiency of stem cell transplantation for infarcted myocardium repair

    Directory of Open Access Journals (Sweden)

    Zhu K

    2014-11-01

    Full Text Available Kai Zhu,1,2 Hao Lai,1,2 Changfa Guo,1,2 Jun Li,1,2 Yulin Wang,1,2 Lingyan Wang,3 Chunsheng Wang1,2 1Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China; 2Shanghai Institute of Cardiovascular Disease, Shanghai, People’s Republic of China; 3Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China Abstract: Mesenchymal stem cell (MSC transplantation has attracted much attention in myocardial infarction therapy. One of the limitations is the poor survival of grafted cells in the ischemic microenvironment. Small interfering RNA-mediated prolyl hydroxylase domain protein 2 (PHD2 silencing in MSCs holds tremendous potential to enhance their survival and paracrine effect after transplantation. However, an efficient and biocompatible PHD2 silencing system for clinical application is lacking. Herein, we developed a novel PHD2 silencing system based on arginine-terminated generation 4 poly(amidoamine (Arg-G4 nanoparticles. The system exhibited effective and biocompatible small interfering RNA delivery and PHD2 silencing in MSCs in vitro. After genetically modified MSC transplantation in myocardial infarction models, MSC survival and paracrine function of IGF-1 were enhanced significantly in vivo. As a result, we observed decreased cardiomyocyte apoptosis, scar size, and interstitial fibrosis, and increased angiogenesis in the diseased myocardium, which ultimately attenuated ventricular remodeling and improved heart function. This work demonstrated that an Arg-G4 nanovector-based PHD2 silencing system could enhance the efficiency of MSC transplantation for infarcted myocardium repair. Keywords: nanoparticles, PHD2, siRNA delivery, mesenchymal stem cells, myocardial infarction

  7. Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity.

    Science.gov (United States)

    Dumoulin, Alexis; Grauschopf, Ulla; Bischoff, Markus; Thöny-Meyer, Linda; Berger-Bächi, Brigitte

    2005-11-01

    DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of -131 mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsbA.

  8. Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA

    Science.gov (United States)

    Grabowska, Anna D.; Wywiał, Ewa; Dunin-Horkawicz, Stanislaw; Łasica, Anna M.; Wösten, Marc M. S. M.; Nagy-Staroń, Anna; Godlewska, Renata; Bocian-Ostrzycka, Katarzyna; Pieńkowska, Katarzyna; Łaniewski, Paweł; Bujnicki, Janusz M.; van Putten, Jos P. M.; Jagusztyn-Krynicka, E. Katarzyna

    2014-01-01

    Background Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. Methods and Results Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. Conclusions Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether

  9. Functional and bioinformatics analysis of two Campylobacter jejuni homologs of the thiol-disulfide oxidoreductase, DsbA.

    Directory of Open Access Journals (Sweden)

    Anna D Grabowska

    Full Text Available BACKGROUND: Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. METHODS AND RESULTS: Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. CONCLUSIONS: Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re

  10. Effects of ara A and fresh medium on chromosome damage and DNA double-strand break repair in X-irradiated stationary cells

    Energy Technology Data Exchange (ETDEWEB)

    Bryant, P.E. (GSF-Abteilung fuer Biophysikalische Strahlenforschung, Frankfurt am Main (Germany, F.R.))

    1984-01-01

    The detailed kinetics of repair of dsb in Ehrlich ascites tumour cells over long repair intervals have been studied and compared under conditions simulating procedures known to cause large changes in cell survival, i.e. holding cells in stationary phase for 7 h after x-radiation, transference of cells to fresh growth medium immediately after x-radiation, and treatment with the DNA synthesis inhibitor 9-..beta..-D-arabinofuranosyladenine (ara A) for 30 min before, during and for 7 h after x-irradiation. These conditions have also been investigated for their effects on frequencies of chromosome abnormalities (anaphase bridges and fragments). Conditions leading to both an inhibition of dsb repair (in the presence of ara A) as well as an acceleration of dsb repair (by fresh growth medium) led to higher frequencies of chromosome abnormalities compared with those for cells under stationary conditions for 7 h after irradiation. Holding dsb open for long periods with ara A may maximize the probability of formation of aberrations, however, the data for fresh medium treatment showed it is not merely the rate at which dsb repair which determines the aberration frequency, and indicated the presence of other biochemical mechanisms in the cell determining the frequency of conversion of dsb into chromosome aberrations.

  11. Phosphorylation: The Molecular Switch of Double-Strand Break Repair

    Directory of Open Access Journals (Sweden)

    K. C. Summers

    2011-01-01

    Full Text Available Repair of double-stranded breaks (DSBs is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ, homologous recombination (HR, or the inclusive DNA damage response (DDR. These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

  12. Knock-in reporter mice demonstrate that DNA repair by non-homologous end joining declines with age.

    Directory of Open Access Journals (Sweden)

    Amita Vaidya

    2014-07-01

    Full Text Available Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs, we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age.

  13. Knock-In Reporter Mice Demonstrate that DNA Repair by Non-homologous End Joining Declines with Age

    Science.gov (United States)

    Vaidya, Amita; Mao, Zhiyong; Tian, Xiao; Spencer, Brianna; Seluanov, Andrei; Gorbunova, Vera

    2014-01-01

    Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs), we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ) pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ) events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age. PMID:25033455

  14. Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.

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    Wilko Duprez

    Full Text Available Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

  15. Smarcal1 promotes double-strand-break repair by nonhomologous end-joining.

    Science.gov (United States)

    Keka, Islam Shamima; Mohiuddin; Maede, Yuko; Rahman, Md Maminur; Sakuma, Tetsushi; Honma, Masamitsu; Yamamoto, Takashi; Takeda, Shunichi; Sasanuma, Hiroyuki

    2015-07-27

    Smarcal1 is a SWI/SNF-family protein with an ATPase domain involved in DNA-annealing activities and a binding site for the RPA single-strand-DNA-binding protein. Although the role played by Smarcal1 in the maintenance of replication forks has been established, it remains unknown whether Smarcal1 contributes to genomic DNA maintenance outside of the S phase. We disrupted the SMARCAL1 gene in both the chicken DT40 and the human TK6 B cell lines. The resulting SMARCAL1(-/-) clones exhibited sensitivity to chemotherapeutic topoisomerase 2 inhibitors, just as nonhomologous end-joining (NHEJ) null-deficient cells do. SMARCAL1(-/-) cells also exhibited an increase in radiosensitivity in the G1 phase. Moreover, the loss of Smarcal1 in NHEJ null-deficient cells does not further increase their radiosensitivity. These results demonstrate that Smarcal1 is required for efficient NHEJ-mediated DSB repair. Both inactivation of the ATPase domain and deletion of the RPA-binding site cause the same phenotype as does null-mutation of Smarcal1, suggesting that Smarcal1 enhances NHEJ, presumably by interacting with RPA at unwound single-strand sequences and then facilitating annealing at DSB ends. SMARCAL1(-/-)cells showed a poor accumulation of Ku70/DNA-PKcs and XRCC4 at DNA-damage sites. We propose that Smarcal1 maintains the duplex status of DSBs to ensure proper recruitment of NHEJ factors to DSB sites. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. On the Jurisdiction of DSB in the Context of the Human Right Law%人权法视野下的 DSB 管辖权

    Institute of Scientific and Technical Information of China (English)

    曾炜; 谭和平

    2014-01-01

    管辖权的确定是任何争端解决的前提条件,对于WTO争端解决机构审理案件来说也概莫能外。通过分析启动WTO争端解决程序的条件和DSB管辖权的特点,指出DSB管辖权是有限的,进而强调DSB不能对人权事项行使管辖权,但这并不妨碍DSB以符合人权法的方式就争议问题作出裁决。%To confirm the jurisdiction is the precondition to resolve any dispute and the DSB of WTO is also not an exception before making a judgment .After analyzing the conditions of initiating the WTO dispute settlement mechanism and the charac-teristics of the jurisdiction of DSB ,the author points out that the jurisdiction of DSB is limited and DSB dose not have jurisdic-tion over the issues which relate to human rights .However ,this does not disturb DSB to make a judgment in conformity with human rights law .

  17. PTH1–34 Blocks Radiation-induced Osteoblast Apoptosis by Enhancing DNA Repair through Canonical Wnt Pathway*

    Science.gov (United States)

    Chandra, Abhishek; Lin, Tiao; Zhu, Ji; Tong, Wei; Huo, Yanying; Jia, Haoruo; Zhang, Yejia; Liu, X. Sherry; Cengel, Keith; Xia, Bing; Qin, Ling

    2015-01-01

    Focal radiotherapy for cancer patients has detrimental effects on bones within the radiation field and the primary clinical signs of bone damage include the loss of functional osteoblasts. We reported previously that daily injection of parathyroid hormone (PTH, 1–34) alleviates radiation-induced osteopenia in a preclinical radiotherapy model by improving osteoblast survival. To elucidate the molecular mechanisms, we irradiated osteoblastic UMR 106-01 cells and calvarial organ culture and demonstrated an anti-apoptosis effect of PTH1–34 on these cultures. Inhibitor assay indicated that PTH exerts its radioprotective action mainly through protein kinase A/β-catenin pathway. γ-H2AX foci staining and comet assay revealed that PTH efficiently promotes the repair of DNA double strand breaks (DSBs) in irradiated osteoblasts via activating the β-catenin pathway. Interestingly, Wnt3a alone also blocked cell death and accelerated DNA repair in primary osteoprogenitors, osteoblastic and osteocytic cells after radiation through the canonical signaling. Further investigations revealed that both Wnt3a and PTH increase the amount of Ku70, a core protein for initiating the assembly of DSB repair machinery, in osteoblasts after radiation. Moreover, down-regulation of Ku70 by siRNA abrogated the prosurvival effect of PTH and Wnt3a on irradiated osteoblasts. In summary, our results identify a novel role of PTH and canonical Wnt signaling in regulating DSB repair machinery and apoptosis in osteoblasts and shed light on using PTH1–34 or Wnt agonist as possible therapy for radiation-induced osteoporosis. PMID:25336648

  18. Recombinational repair of radiation-induced double-strand breaks occurs in the absence of extensive resection

    Science.gov (United States)

    Westmoreland, James W.; Resnick, Michael A.

    2016-01-01

    Recombinational repair provides accurate chromosomal restitution after double-strand break (DSB) induction. While all DSB recombination repair models include 5′-3′ resection, there are no studies that directly assess the resection needed for repair between sister chromatids in G-2 arrested cells of random, radiation-induced ‘dirty’ DSBs. Using our Pulse Field Gel Electrophoresis-shift approach, we determined resection at IR-DSBs in WT and mutants lacking exonuclease1 or Sgs1 helicase. Lack of either reduced resection length by half, without decreased DSB repair or survival. In the exo1Δ sgs1Δ double mutant, resection was barely detectable, yet it only took an additional hour to achieve a level of repair comparable to WT and there was only a 2-fold dose-modifying effect on survival. Results with a Dnl4 deletion strain showed that remaining repair was not due to endjoining. Thus, similar to what has been shown for a single, clean HO-induced DSB, a severe reduction in resection tract length has only a modest effect on repair of multiple, dirty DSBs in G2-arrested cells. Significantly, this study provides the first opportunity to directly relate resection length at DSBs to the capability for global recombination repair between sister chromatids. PMID:26503252

  19. A mediator methylation mystery: JMJD1C demethylates MDC1 to regulate DNA repair.

    Science.gov (United States)

    Lu, Jian; Matunis, Michael J

    2013-12-01

    Mediator of DNA-damage checkpoint 1 (MDMDC1) has a central role in repair of DNA double-strand breaks (DSBs) by both homologous recombination and nonhomologous end joining, and its function is regulated by post-translational phosphorylation, ubiquitylation and sumoylation. In this issue, a new study by Watanabe et al. reveals that methylation of MDMDC1 is also critical for its function in DSB repair and specifically affects repair through BRCA1-dependent homologous recombination.

  20. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  1. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  2. Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

    Energy Technology Data Exchange (ETDEWEB)

    Stenerl& #246; w, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn; Stenerlow, Bo

    2008-04-29

    Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.

  3. A receiver function study across the Dead Sea Basin (DSB)

    Science.gov (United States)

    Mohsen, A.; Asch, G.; Hofstetter, R.; Kind, R.; Weber, M.

    2008-12-01

    Beginning in September 2006, a temporary network of 30 broadband and 45 short-period seismic stations has been set up on both sides of the Dead Sea Basin (DSB). During one and a half year of successful operation, data were continuously recorded in the field at 100 Hz and 200 Hz sample frequency for the broadband and short-period seismic stations, respectively. The raw data were converted to miniseed format and archived as full seed volume in the GEOFON data center of the GFZ. In the present work, the Receiver Function Method has been applied to the three component passive source data to investigate seismic discontinuities from the crust down to the upper mantle. Unusual negative phases at about 1s delay time have been observed at several stations in the Dead Sea region on the top of the assumed salt diapir. First preliminary receiver function analysis reveals a crustal thickness of about 30 -35 km in the investigated area and possibly low-velocity layer beneath the Moho. It also shows a basin which is possibly filled with salt about 10 km thick beneath the Lisan peninsula (Dead Sea).

  4. Blockage of Autophagy in C6 Glioma Cells Enhanced Radiosensitivity Possibly by Attenuating DNA-PK-Dependent DSB Due to Limited Ku Nuclear Translocation and DNA Binding.

    Science.gov (United States)

    Liu, C; He, W; Jin, M; Li, H; Xu, H; Liu, H; Yang, K; Zhang, T; Wu, G; Ren, J

    2015-01-01

    Glioblastoma multiforme (GBM) is the most lethal brain tumor and notorious for its resistance to ionizing radiation (IR). Recent evidence suggests that one possible mechanism that enables resistance to IR and protects cells against therapeutic stress is cellular autophagy. The molecular basis for this pro-survival function, however, remains elusive. Herein, we report a molecular mechanism by which IR-induced autophagy accelerates the repair of DNA double-strand breaks (DSB). We demonstrate that IR induces the accumulation of autophagosomes, which is accompanied by elevated expression of autophagyrelated genes beclin-1, atg5, atg7, and atg12. Beclin-1 knockdown impaired the induction of IR-mediated autophagy and significantly sensitized glioma cells to radiation therapy in vitro and in vivo. Furthermore, our data is the first to demonstrate that the radiosensitizing effect of beclin-1 knockdown may result from the disruption of nuclear translocation and DNA binding activity of Ku proteins and consequent attenuation of DSB repair. Our findings help advance our understanding of the molecular mechanisms underlying IR-induced autophagy and provide a promising adjunctive therapeutic strategy for the radiosensitization of malignant glioma.

  5. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, D.J.C.; Stackhouse, M. [Los Alamos National Lab., NM (United States); Chen, D.S. [Rochester Univ., NY (United States). Dept. of Radiation Oncology

    1993-02-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  6. DNA repair and radiation sensitivity in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, D.J.C.; Stackhouse, M. (Los Alamos National Lab., NM (United States)); Chen, D.S. (Rochester Univ., NY (United States). Dept. of Radiation Oncology)

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population.

  7. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.

    Directory of Open Access Journals (Sweden)

    Nicole Bennardo

    2008-06-01

    Full Text Available Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ, single-strand annealing (SSA, and homology directed repair (HDR/GC. Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI-induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.

  8. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.

    Directory of Open Access Journals (Sweden)

    Nicole Bennardo

    2008-06-01

    Full Text Available Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ, single-strand annealing (SSA, and homology directed repair (HDR/GC. Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI-induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.

  9. Repair of DNA Double-Strand Breaks

    Science.gov (United States)

    Falk, Martin; Lukasova, Emilie; Kozubek, Stanislav

    The genetic information of cells continuously undergoes damage induced by intracellular processes including energy metabolism, DNA replication and transcription, and by environmental factors such as mutagenic chemicals and UV and ionizing radiation. This causes numerous DNA lesions, including double strand breaks (DSBs). Since cells cannot escape this damage or normally function with a damaged genome, several DNA repair mechanisms have evolved. Although most "single-stranded" DNA lesions are rapidly removed from DNA without permanent damage, DSBs completely break the DNA molecule, presenting a real challenge for repair mechanisms, with the highest risk among DNA lesions of incorrect repair. Hence, DSBs can have serious consequences for human health. Therefore, in this chapter, we will refer only to this type of DNA damage. In addition to the biochemical aspects of DSB repair, which have been extensively studied over a long period of time, the spatio-temporal organization of DSB induction and repair, the importance of which was recognized only recently, will be considered in terms of current knowledge and remaining questions.

  10. An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Mirta M L Sousa

    Full Text Available Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs. Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ pathway of double-strand break (DSB repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel

  11. Transcriptional regulation of the assT-dsbL-dsbI gene cluster in Salmonella enterica serovar Typhi IMSS-1 depends on LeuO, H-NS, and specific growth conditions.

    Science.gov (United States)

    Gallego-Hernández, A L; Hernández-Lucas, I; De la Cruz, M A; Olvera, L; Morett, E; Medina-Aparicio, L; Ramírez-Trujillo, J A; Vázquez, A; Fernández-Mora, M; Calva, E

    2012-05-01

    The assT gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In Salmonella enterica serovar Typhi IMSS-1, the assT gene is located upstream of the dsbL and dsbI genes, which are involved in a disulfide bond formation required for its activation. The assT-dsbL-dsbI gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned leuO and in a ΔleuO background, two transcription start sites were detected for assT and two for dsbL-dsbI in minimal medium. The H-NS nucleoid protein repressed the expression of the assT-dsbL-dsbI LeuO-dependent operon, as well as of the assT transcriptional units. Thus, the expression of the assT-dsbL-dsbI gene cluster depends on the global regulatory proteins LeuO and H-NS, as well as on specific growth conditions.

  12. Radio-over-fiber DSB-to-SSB conversion using semiconductor lasers at stable locking dynamics.

    Science.gov (United States)

    Hsieh, Kun-Lin; Hung, Yu-Han; Hwang, Sheng-Kwang; Lin, Chien-Chung

    2016-05-01

    In radio-over-fiber systems, optical single-sideband (SSB) modulation signals are preferred to optical double-sideband (DSB) modulation signals for fiber distribution in order to mitigate the microwave power fading effect. However, typically adopted modulation schemes generate DSB signals, making DSB-to-SSB conversion necessary before or after fiber distribution. This study investigates a semiconductor laser at stable locking dynamics for such conversion. The conversion relies solely on the nonlinear dynamical interaction between an input DSB signal and the laser. Only a typical semiconductor laser is therefore required as the key conversion unit, and no pump or probe signal is necessary. The conversion can be achieved for a broad tunable range of microwave frequency up to at least 60 GHz. In addition, the conversion can be carried out even when the microwave frequency, the power of the input DSB signal, or the frequency of the input DSB signal fluctuates over a wide range, leading to high adaptability and stability of the conversion system. After conversion, while the microwave phase quality, such as linewidth and phase noise, is mainly preserved, a bit-error ratio down to 10-9 is achieved for a data rate up to at least 8 Gb/s with a detection sensitivity improvement of more than 1.5 dB.

  13. Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis

    Energy Technology Data Exchange (ETDEWEB)

    Kurz, M. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia); Iturbe-Ormaetxe, I. [School of Integrative Biology, The University of Queensland, St Lucia, QLD 4072 (Australia); Jarrott, R. [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia); O’Neill, S. L. [School of Integrative Biology, The University of Queensland, St Lucia, QLD 4072 (Australia); Byriel, K. A.; Martin, J. L., E-mail: j.martin@imb.uq.edu.au; Heras, B., E-mail: j.martin@imb.uq.edu.au [Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, St Lucia, QLD 4072 (Australia)

    2008-02-01

    The first crystallization of a W. pipientis protein, α-DsbA1, was achieved using hanging-drop and sitting-drop vapour diffusion. α-DsbA1 is one of two DsbA homologues encoded by the Gram-negative α-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The α-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet α-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, b = 49.5, c = 69.3 Å, β = 107.0° and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 Å using a copper anode and data from SeMet α-DsbA1 crystals were recorded to 2.45 Å resolution using a chromium anode.

  14. Search for novel remedies to augment radiation resistance of inhabitants of Fukushima and Chernobyl disasters: identifying DNA repair protein XRCC4 inhibitors.

    Science.gov (United States)

    Sun, Mao-Feng; Chen, Hsin-Yi; Tsai, Fuu-Jen; Lui, Shu-Hui; Chen, Chih-Yi; Chen, Calvin Yu-Chian

    2011-10-01

    Two nuclear plant disasters occurring within a span of 25 years threaten health and genome integrity both in Fukushima and Chernobyl. Search for remedies capable of enhancing DNA repair efficiency and radiation resistance in humans appears to be a urgent problem for now. XRCC4 is an important enhancer in promoting repair pathway triggered by DNA double-strand break (DSB). In the context of radiation therapy, active XRCC4 could reduce DSB-mediated apoptotic effect on cancer cells. Hence, developing XRCC4 inhibitors could possibly enhance radiotherapy outcomes. In this study, we screened traditional Chinese medicine (TCM) database, TCM Database@Taiwan, and have identified three potent inhibitor agents against XRCC4. Through molecular dynamics simulation, we have determined that the protein-ligand interactions were focused at Lys188 on chain A and Lys187 on chain B. Intriguingly, the hydrogen bonds for all three ligands fluctuated frequently but were held at close approximation. The pi-cation interactions and ionic interactions mediated by o-hydroxyphenyl and carboxyl functional groups respectively have been demonstrated to play critical roles in stabilizing binding conformations. Based on these results, we reported the identification of potential radiotherapy enhancers from TCM. We further characterized the key binding elements for inhibiting the XRCC4 activities.

  15. Extremophilic Acinetobacter Strains from High-Altitude Lakes in Argentinean Puna: Remarkable UV-B Resistance and Efficient DNA Damage Repair

    Science.gov (United States)

    Albarracín, Virginia Helena; Pathak, Gopal P.; Douki, Thierry; Cadet, Jean; Borsarelli, Claudio Darío; Gärtner, Wolfgang; Farias, María Eugenia

    2012-06-01

    High-Altitude Andean Lakes (HAAL) of the South American Andes are almost unexplored ecosystems of shallow lakes. The HAAL are recognized by a remarkably high UV exposure, strong changes in temperature and salinity, and a high content of toxic elements, especially arsenic. Being exposed to remarkably extreme conditions, they have been classified as model systems for the study of life on other planets. Particularly, Acinetobacter strains isolated from the HAAL were studied for their survival competence under strong UV-B irradiation. Clinical isolates, Acinetobacter baumannii and Acinetobacter johnsonii, served as reference material. Whereas the reference strains rapidly lost viability under UV-B irradiation, most HAAL-derived strains readily survived this exposure and showed less change in cell number after the treatment. Controls for DNA repair activity, comparing dark repair (DR) or photo repair (PR), gave evidence for the involvement of photolyases in the DNA repair. Comparative measurements by HPLC-mass spectrometry detected the number of photoproducts: bipyrimidine dimers under both PR and DR treatments were more efficiently repaired in the HAAL strains (up to 85 % PR and 38 % DR) than in the controls (31 % PR and zero DR ability). Analysis of cosmid-cloned total genomic DNA from the most effective DNA-photorepair strain (Ver3) yielded a gene (HQ443199) encoding a protein with clear photolyase signatures belonging to class I CPD-photolyases. Despite the relatively low sequence similarity of 41 % between the enzymes from Ver3 and from E. coli (PDB 1DNPA), a model-building approach revealed a high structural homology to the CPD-photolyase of E. coli.

  16. Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

    Science.gov (United States)

    Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C; Glickman, Michael S

    2014-08-01

    Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis.

  17. DSB:Ce3+ scintillation glass for future

    Science.gov (United States)

    Auffray, E.; Akchurin, N.; Benaglia, A.; Borisevich, A.; Cowden, C.; Damgov, J.; Dormenev, V.; Dragoiu, C.; Dudero, P.; Korjik, M.; Kozlov, D.; Kunori, S.; Lecoq, P.; Lee, S. W.; Lucchini, M.; Mechinsky, V.; Pauwels, K.

    2015-02-01

    One of the main challenges for detectors at future high-energy collider experiments is the high precision measurement of hadron and jet energy and momentum. One possibility to achieve this is the dual-readout technique, which allows recording simultaneously scintillation and Cherenkov light in an active medium in order to extract the electromagnetic fraction of the total shower energy on an event- by-event basis. Making use of this approach in the high luminosity LHC, however, puts stringent requirements on the active materials in terms of radiation hardness. Consequently, the R&D carried out on suitable scintillating materials focuses on the detector performance as well as on radiation tolerance. Among the different scintillating materials under study, scintillating glasses can be a suitable solution due to their relatively simple and cost effective production. Recently a new type of inorganic scintillating glass: Cerium doped DSB has been developed by Radiation Instruments and New Components LLC in Minsk for oil logging industry. This material can be produced either in form of bulk or fiber shape with diameter 0.3-2mm and length up to 2000 mm. It is obtained by standard glass production technology at temperature 1400°C with successive thermal annealing treatment at relatively low temperature. The production of large quantities is relatively easy and the production costs are significantly lower compared to crystal fibers. Therefore, this material is considered as an alternative and complementary solution to crystal fibers in view of a production at industrial scale, as required for a large dual readout calorimeter. In this paper, the first results on optical, scintillation properties as well as the radiation damage behaviour obtained on different samples made with different raw materials and various cerium concentrations will be presented.

  18. Legal Suggestions for Enforcement of DSB Reports in China%中国执行 DSB 裁决报告的法律对策和建议

    Institute of Scientific and Technical Information of China (English)

    李晓郛; 纪演娟

    2014-01-01

    When enforcing the DSB reports , there are two questions in front of China:one is that the DSB reports are related with many fields and the other is the difficulty of execution of DSB reports .As the most important member in WTO , US inclines to use pragmatism and considers the national interest as the start-ing point for execution of DSB reports .Harmonizing system which talkes USTR as the main point is con-sidered as a main characteristic .Union integration is the main reason if EU would like to carry out DSB reports .Based on Chinese conditions , combined with the practice of the most WTO members , there is no need to give DSB reports direct effect .The paper recommends China establish the general idea of the su-premacy of national interests and a coordination system with a committee of experts .Meanwhile , the do-mestic courts take judicial restraints when confronting DSB reports and related litigations .%执行所涉领域广、执行难度大是中国执行DSB裁决报告的两个主要难题。作为WTO最重要的成员方,美国以实用主义为先导、国家利益为出发点,执行DSB裁决报告,以USTR为主的协调机制亦是其一大特色;欧盟以有利于联盟一体化的态度执行DSB裁决报告。立足中国国情,结合WTO成员方的执行实践,建议今后在执行DSB裁决报告时,树立国家利益至上的总体思路,建立协调人制度,并设立专家委员会配合执行,国内法院以司法克制的态度处理DSB裁决报告及相关诉讼。

  19. Crustal structure of the Dead Sea Basin (DSB) from a receiver function analysis

    Science.gov (United States)

    Mohsen, A.; Asch, G.; Mechie, J.; Kind, R.; Hofstetter, R.; Weber, M.; Stiller, M.; Abu-Ayyash, K.

    2011-01-01

    The Dead Sea Transform (DST) is a major left-lateral strike-slip fault that accommodates the relative motion between the African and Arabian plates, connecting a region of extension in the Red Sea to the Taurus collision zone in Turkey over a length of about 1100 km. The Dead Sea Basin (DSB) is one of the largest basins along the DST. The DSB is a morphotectonic depression along the DST, divided into a northern and a southern sub-basin, separated by the Lisan salt diapir. We report on a receiver function study of the crust within the multidisciplinary geophysical project, DEad Sea Integrated REsearch (DESIRE), to study the crustal structure of the DSB. A temporary seismic network was operated on both sides of the DSB between 2006 October and 2008 April. The aperture of the network is approximately 60 km in the E-W direction crossing the DSB on the Lisan peninsula and about 100 km in the N-S direction. Analysis of receiver functions from the DESIRE temporary network indicates that Moho depths vary between 30 and 38 km beneath the area. These Moho depth estimates are consistent with results of near-vertical incidence and wide-angle controlled-source techniques. Receiver functions reveal an additional discontinuity in the lower crust, but only in the DSB and west of it. This leads to the conclusion that the internal crustal structure east and west of the DSB is different at the present-day. However, if the 107 km left-lateral movement along the DST is taken into account, then the region beneath the DESIRE array where no lower crustal discontinuity is observed would have lain about 18 Ma ago immediately adjacent to the region under the previous DESERT array west of the DST where no lower crustal discontinuity is recognized.

  20. Development of novel visual-plus quantitative analysis systems for studying DNA double-strand break repairs in zebrafish.

    Science.gov (United States)

    Liu, Jingang; Gong, Lu; Chang, Changqing; Liu, Cong; Peng, Jinrong; Chen, Jun

    2012-09-20

    The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.

  1. Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

    Institute of Scientific and Technical Information of China (English)

    Jingang Liu; Lu Gong; Changqing Chang; Cong Liu; Jinrong Peng; Jun Chen

    2012-01-01

    The use of reporter systems to analyze DNA double-strand break (DSB) repairs,based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce Ⅰ,is usually carried out with cell lines.In this study,we developed three visual-plus quantitative assay systems for homologous recombination (HR),non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos.To initiate DNA DSB repair,we used two I-Sce Ⅰ recognition sites in opposite orientation rather than the usual single site.The NHEJ,HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions,and the repair of DNA lesion caused by I-Sce Ⅰ could be tracked by EGFP expression in the embryos.Apart from monitoring the intensity of green fluorescence,the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR).Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos.Furthermore,while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52,respectively,NHEJ could only be impaired by the knockdown of ligaseⅣ (lig4) when the NHEJ construct was cut by I-Sce Ⅰ in vivo.More interestingly,blocking NHEJ with lig4-MO increased the frequency of HR,but decreased the frequency of SSA.Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal,and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.

  2. Transcriptional Regulation of the assT-dsbL-dsbI Gene Cluster in Salmonella enterica Serovar Typhi IMSS-1 Depends on LeuO, H-NS, and Specific Growth Conditions

    OpenAIRE

    Gallego-Hernández, A. L.; Hernández-Lucas, I.; De la Cruz, M. A.; Olvera, L.; Morett, E; Medina-Aparicio, L.; Ramírez-Trujillo, J. A.; Vázquez, A.; Fernández-Mora, M.; Calva, E

    2012-01-01

    The assT gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In Salmonella enterica serovar Typhi IMSS-1, the assT gene is located upstream of the dsbL and dsbI genes, which are involved in a disulfide bond formation required for its activation. The assT-dsbL-dsbI gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned leuO and in a Δ...

  3. Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases.

    Science.gov (United States)

    Kocher, Thomas; Peking, Patricia; Klausegger, Alfred; Murauer, Eva Maria; Hofbauer, Josefina Piñón; Wally, Verena; Lettner, Thomas; Hainzl, Stefan; Ablinger, Michael; Bauer, Johann Wolfgang; Reichelt, Julia; Koller, Ulrich

    2017-08-24

    With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Nonlinear Dynamics of Photonics for Optical Signal Processing - Optical Frequency Conversion and Optical DSB-to-SSB Conversion

    Science.gov (United States)

    2015-09-17

    processing - optical frequency conversion and optical DSB -to-SSB conversion 5a. CONTRACT NUMBER FA2386-14-1-0006 5b. GRANT NUMBER Grant 134113...nonlinear dynamics of semiconductor lasers for certain optical signal processing functionalities, including optical DSB -to-SSB conversion, photonic...conversion and optical DSB -to-SSB conversion Performance Period May 30, 2014 ~ May 29, 2015 Principal Investigator Name: Sheng-Kwang Hwang Position

  5. Novel Image Processing Interface to Relate DSB Spatial Distribution from Immunofluorescence Foci Experiments to the State-of-the-Art Models of DNA Breakage

    Science.gov (United States)

    Ponomarev, A. L.; Cucinotta, F. A.

    2004-01-01

    A recently developed software (NASARadiationTrackImage) allows a quick and automatic segmentation of foci that indicate spatial localization of specific proteins that are visualized by immunofluorescence. Of interest are the spatial and temporal distribution of foci such as gammaH2AX, a signal of the phosphorylation of a variant of the histone H2A that has been shown to correspond to DSBs, or proteins involved in DSB processing, such as ATM, Rad51, and p53, following exposures of human cells to high charge and energy (HZE) ion irradiation. Experimental data are recorded as sets of two-dimensional images in color with cells and foci of gammaH2AX, ATM, Rad51 or others shown. Different cells, levels of radiation and timing after radiation were recorded. The software allows us to calculate the number of foci per cell, overall intensity of light in foci and their spatial organization. A simple statistical model allows for testing of foci overlap (eclipse). A more complex statistical model previously known as DNAbreak simulates track structure and random chromosome geometry. It has one adjustable parameter corresponding to an average intensity of DSB creation in cubic micrometers of DNA volume per particle track or unit dose. Its limitation is the low-resolution limit both in physical space and DSB's along DNA. It works adequately on the scale of a cell and provides further insights on how the geometry of tracks and DNA affects genomic damage of the cell and subsequent repair. Future developments of the model for the description of the time evolution of DNA damage response proteins, and more robust track structure models will be discussed.

  6. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ivanov, E L; Haber, J E

    1995-04-01

    HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.

  7. Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells.

    Science.gov (United States)

    Bindra, Ranjit S; Goglia, Alexander G; Jasin, Maria; Powell, Simon N

    2013-06-01

    Double-strand break (DSB) repair pathways are critical for the maintenance of genomic integrity and the prevention of tumorigenesis in mammalian cells. Here, we present the development and validation of a novel assay to measure mutagenic non-homologous end-joining (NHEJ) repair in living cells, which is inversely related to canonical NHEJ and is based on the sequence-altering repair of a single site-specific DSB at an intrachromosomal locus. We have combined this mutagenic NHEJ assay with an established homologous recombination (HR) assay such that both pathways can be monitored simultaneously. In addition, we report the development of a ligand-responsive I-SceI protein, in which the timing and kinetics of DSB induction can be precisely controlled by regulating protein stability and cellular localization in cells. Using this system, we report that mutagenic NHEJ repair is suppressed in growth-arrested and serum-deprived cells, suggesting that end-joining activity in proliferating cells is more likely to be mutagenic. Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.

  8. Functional diversity of three different DsbA proteins from Neisseria meningitidis.

    Science.gov (United States)

    Sinha, Sunita; Langford, Paul R; Kroll, J Simon

    2004-09-01

    The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes - nmb0278, nmb0294 and nmb0407 - encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.

  9. DNA repair by RNA: Templated, or not templated, that is the question.

    Science.gov (United States)

    Meers, Chance; Keskin, Havva; Storici, Francesca

    2016-08-01

    Cells are continuously exposed to both endogenous and exogenous sources of genomic stress. To maintain chromosome stability, a variety of mechanisms have evolved to cope with the multitude of genetic abnormalities that can arise over the life of a cell. Still, failures to repair these lesions are the driving force of cancers and other degenerative disorders. DNA double-strand breaks (DSBs) are among the most toxic genetic lesions, inhibiting cell ability to replicate, and are sites of mutations and chromosomal rearrangements. DSB repair is known to proceed via two major mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR reliance on the exchange of genetic information between two identical or nearly identical DNA molecules offers increased accuracy. While the preferred substrate for HR in mitotic cells is the sister chromatid, this is limited to the S and G2 phases of the cell cycle. However, abundant amounts of homologous genetic substrate may exist throughout the cell cycle in the form of RNA. Considered an uncommon occurrence, the direct transfer of information from RNA to DNA is thought to be limited to special circumstances. Studies have shown that RNA molecules reverse transcribed into cDNA can be incorporated into DNA at DSB sites via a non-templated mechanism by NHEJ or a templated mechanism by HR. In addition, synthetic RNA molecules can directly template the repair of DSBs in yeast and human cells via an HR mechanism. New work suggests that even endogenous transcript RNA can serve as a homologous template to repair a DSB in chromosomal DNA. In this perspective, we will review and discuss the recent advancements in DSB repair by RNA via non-templated and templated mechanisms. We will provide current findings, models and future challenges investigating RNA and its role in DSB repair.

  10. Chromatin modification and NBS1: their relationship in DNA double-strand break repair.

    Science.gov (United States)

    Saito, Yuichiro; Zhou, Hui; Kobayashi, Junya

    2016-01-01

    The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

  11. Orphan receptor TR3 enhances p53 transactivation and represses DNA double-strand break repair in hepatoma cells under ionizing radiation.

    Science.gov (United States)

    Zhao, Bi-xing; Chen, Hang-zi; Du, Xiao-dan; Luo, Jie; He, Jian-ping; Wang, Rong-hao; Wang, Yuan; Wu, Rong; Hou, Ru-rong; Hong, Ming; Wu, Qiao

    2011-08-01

    In response to ionizing radiation (IR)-induced DNA double-strand breaks (DSB), cells elicit an evolutionarily conserved checkpoint response that induces cell cycle arrest and either DNA repair or apoptosis, thereby maintaining genomic stability. DNA-dependent protein kinase (DNA-PK) is a central enzyme involved in DSB repair for mammalian cells that comprises a DNA-PK catalytic subunit and the Ku protein, which act as regulatory elements. DNA-PK also functions as a signaling molecule to selectively regulate p53-dependent apoptosis in response to IR. Herein, we demonstrate that the orphan nuclear receptor TR3 suppresses DSB repair by blocking Ku80 DNA-end binding activity and promoting DNA-PK-induced p53 activity in hepatoma cells. We find that TR3 interacts with Ku80 and inhibits its binding to DNA ends, which then suppresses DSB repair. Furthermore, TR3 is a phosphorylation substrate for DNA-PK and interacts with DNA-PK catalytic subunit in a Ku80-independent manner. Phosphorylated TR3, in turn, enhances DNA-PK-induced phosphorylation and p53 transcription activity, thereby enhancing IR-induced apoptosis in hepatoma cells. Together, our findings reveal novel functions for TR3, not only in DSB repair regulation but also in IR-induced hepatoma cell apoptosis, and they suggest that TR3 is a potential target for cancer radiotherapy.

  12. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks.

    Science.gov (United States)

    Van Meter, Michael; Simon, Matthew; Tombline, Gregory; May, Alfred; Morello, Timothy D; Hubbard, Basil P; Bredbenner, Katie; Park, Rosa; Sinclair, David A; Bohr, Vilhelm A; Gorbunova, Vera; Seluanov, Andrei

    2016-09-06

    The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6), promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB) repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  13. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

    Directory of Open Access Journals (Sweden)

    Michael Van Meter

    2016-09-01

    Full Text Available The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6, promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK, phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose polymerase 1 (PARP1 to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  14. Phosphatase complex Pph3/Psy2 is involved in regulation of efficient non-homologous end-joining pathway in the yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Katayoun Omidi

    Full Text Available One of the main mechanisms for double stranded DNA break (DSB repair is through the non-homologous end-joining (NHEJ pathway. Using plasmid and chromosomal repair assays, we showed that deletion mutant strains for interacting proteins Pph3p and Psy2p had reduced efficiencies in NHEJ. We further observed that this activity of Pph3p and Psy2p appeared linked to cell cycle Rad53p and Chk1p checkpoint proteins. Pph3/Psy2 is a phosphatase complex, which regulates recovery from the Rad53p DNA damage checkpoint. Overexpression of Chk1p checkpoint protein in a parallel pathway to Rad53p compensated for the deletion of PPH3 or PSY2 in a chromosomal repair assay. Double mutant strains Δpph3/Δchk1 and Δpsy2/Δchk1 showed additional reductions in the efficiency of plasmid repair, compared to both single deletions which is in agreement with the activity of Pph3p and Psy2p in a parallel pathway to Chk1p. Genetic interaction analyses also supported a role for Pph3p and Psy2p in DNA damage repair, the NHEJ pathway, as well as cell cycle progression. Collectively, we report that the activity of Pph3p and Psy2p further connects NHEJ repair to cell cycle progression.

  15. A role for small RNAs in DNA double-strand break repair

    DEFF Research Database (Denmark)

    Wei, W.; Ba, Z.; Wu, Y.

    2012-01-01

    Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cel...

  16. Have a break: determinants of meiotic DNA double strand break (DSB) formation and processing in plants.

    Science.gov (United States)

    Edlinger, Bernd; Schlögelhofer, Peter

    2011-03-01

    Meiosis is an essential process for sexually reproducing organisms, leading to the formation of specialized generative cells. This review intends to highlight current knowledge of early events during meiosis derived from various model organisms, including plants. It will particularly focus on cis- and trans-requirements of meiotic DNA double strand break (DSB) formation, a hallmark event during meiosis and a prerequisite for recombination of genetic traits. Proteins involved in DSB formation in different organisms, emphasizing the known factors from plants, will be introduced and their functions outlined. Recent technical advances in DSB detection and meiotic recombination analysis will be reviewed, as these new tools now allow analysis of early meiotic recombination in plants with incredible accuracy. To anticipate future directions in plant meiosis research, unpublished results will be included wherever possible.

  17. Effects of cadmium on nuclear integrity and DNA repair efficiency in the gill cells of Mytilus edulis L.

    Science.gov (United States)

    Pruski, Audrey M; Dixon, David R

    2002-05-01

    Although the effects of heavy metals on marine invertebrate species are well studied in term of their toxicity and bioaccumulation, less is known about their genotoxicity. The aim of this investigation was to assess the DNA damaging potential of cadmium (Cd) in an important pollution sentinel organism, the mussel Mytilus edulis. Cadmium is one of the most toxic and widespread heavy metals found in the marine environment, and is a recognised carcinogen in mammals. Based on the results of the comet assay (alkaline single cell gel electrophoresis), Cd was found not to be genotoxic in mussel gill cells under acute and chronic exposure conditions, whereas pre-exposure to low concentrations of Cd was found to enhance the genotoxicity of another mutagen, hydrogen peroxide (H2O2). The effects of H2O2 were normally reversible when cells were transferred to clean saline buffer. However, in cells that had been pre-treated with Cd, in vivo or in vitro, we observed a decrease in this post-treatment DNA repair. The effects of Cd were reversed by zinc which suggests that the inhibitory effect of Cd on DNA repair was due to the displacement of zinc ions from active sites on proteins involved in the repair process (a property already described for mammals). Moreover, since Cd inhibits or delays the onset of apoptosis (programmed cell death), this removes one of the main defence mechanisms responsible for protecting the organism against neoplasia. There appears to be a close similarity between the effects of Cd on marine molluscs and mammals.

  18. ERCC2/XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway.

    Science.gov (United States)

    Zhang, Guopei; Guan, Yangyang; Zhao, Yuejiao; van der Straaten, Tahar; Xiao, Sha; Xue, Ping; Zhu, Guolian; Liu, Qiufang; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Lu, Xiaobo

    2017-02-01

    Platinum-based treatment causes Pt-DNA adducts which lead to cell death. The platinum-induced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system of which ERCC2/XPD is a critical enzyme. Single nucleotide polymorphisms in ERCC2/XPD have been found to be associated with platinum resistance. The aim of the present study was to investigate whether ERCC2/XPD Lys751Gln (rs13181) polymorphism is causally related to DNA repair capacity of platinum-induced DNA damage. First, cDNA clones expressing different genotypes of the polymorphism was transfected to an ERCC2/XPD defective CHO cell line (UV5). Second, all cells were treated with cisplatin. Cellular survival rate were investigated by MTT growth inhibition assay, DNA damage levels were investigated by comet assay and RAD51 staining. The distribution of cell cycle and the change of apoptosis rates were detected by a flow cytometric method (FCM). Finally, P53mRNA and phospho-P53 protein levels were further investigated in order to explore a possible explanation. As expected, there was a significantly increased in viability of UV5(ERCC2 (AA)) as compared to UV5(ERCC2 (CC)) after cisplatin treatment. The DNA damage level of UV5(ERCC2 (AA)) was significant decreased compared to UV5(ERCC2 (CC)) at 24 h of treatment. Mutation of ERCC2rs13181 AA to CC causes a prolonged S phase in cell cycle. UV5(ERCC2 (AA)) alleviated the apoptosis compared to UV5(ERCC2 (CC)), meanwhile P53mRNA levels in UV(ERCC2 (AA)) was also lower when compared UV5(ERCC2 (CC)). It co-incides with a prolonged high expression of phospho-P53, which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). We concluded that ERCC2/XPD rs13181 polymorphism is possibly related to the DNA repair capacity of platinum-induced DNA damage. This functional study provides some clues to clarify the relationship between cisplatin resistance and ERCC2/XPDrs13181 polymorphism.

  19. Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining.

    Directory of Open Access Journals (Sweden)

    Richard Bowater

    2006-02-01

    Full Text Available DNA double-strand breaks (DSBs are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ. Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.

  20. Regulation of DNA double-strand break repair pathway choice

    Institute of Scientific and Technical Information of China (English)

    Meena Shrivastav; Leyma P De Haro; Jac A Nickoloff

    2008-01-01

    DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources includ-ing reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1 (XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.

  1. Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

    Energy Technology Data Exchange (ETDEWEB)

    Premkumar, Lakshmanane, E-mail: p.lakshmanane@imb.uq.edu.au; Heras, Begoña; Duprez, Wilko; Walden, Patricia; Halili, Maria; Kurth, Fabian; Fairlie, David P.; Martin, Jennifer L., E-mail: p.lakshmanane@imb.uq.edu.au [University of Queensland, St Lucia, QLD 4067 (Australia)

    2013-10-01

    The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics. The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited

  2. Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining

    OpenAIRE

    Bowater, Richard; Doherty, Aidan J.

    2006-01-01

    DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHE...

  3. DNA double strand break repair via non-homologous end-joining

    OpenAIRE

    Davis, Anthony J.; Chen, David J.

    2013-01-01

    DNA double-stranded breaks (DSB) are among the most dangerous forms of DNA damage. Unrepaired DSBs results in cells undergoing apoptosis or senescence whereas mis-processing of DSBs can lead to genomic instability and carcinogenesis. One important pathway in eukaryotic cells responsible for the repair of DSBs is non-homologous end-joining (NHEJ). In this review we will discuss the interesting new insights into the mechanism of the NHEJ pathway and the proteins which mediate this repair proces...

  4. Peroxisome proliferator-activated receptor γ enhances adiponectin secretion via up-regulating DsbA-L expression.

    Science.gov (United States)

    Jin, Dan; Sun, Jun; Huang, Jing; Yu, Xiaoling; Yu, An; He, Yiduo; Li, Qiang; Yang, Zaiqing

    2015-08-15

    Disulfide-bond A oxidoreductase like-protein (DsbA-L) was identified as a molecular chaperone facilitating the assembly and secretion of adiponectin, an adipokine with multiple beneficial effects. In obesity the level of DsbA-L is reduced with a concomitant decrease of the circulating adiponectin level, especially of the high molecular weight form (HMW). Both rodent and human studies have shown that the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-γ agonists increase adiponectin levels in serum by activating PPARγ, which up-regulates critical endoplasmic reticulum (ER) chaperones thus facilitating protein folding. As shown in the present study, overexpression of PPARγ in human embryonic kidney (HEK) 293 cells elicited the cellular release of HMW adiponectin. PPARγ enhanced expression of DsbA-L by binding directly to peroxisome proliferator response element (PPRE) site within the DsbA-L promoter. Conversely, in differentiated 3T3-L1 cells, PPARγ knockdown resulted in decreased expression of Adiponectin, DsbA-L and ERp44. DsbA-L expression increased after PPARγ agonist treatment and decreased upon treatment with PPARγ antagonist in 3T3-L1 adipocytes. DsbA-L deficiency in differentiated 3T3-L1 cells impaired the secretion of adiponectin. We therefore propose that DsbA-L plays an important role in facilitating HMW adiponectin formation and release from cells under the regulation of PPARγ. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Functional and bioinformatics analysis of two Campylobacter jejuni homologs of the thiol-disulfide oxidoreductase, DsbA

    NARCIS (Netherlands)

    Grabowska, Anna D; Wywiał, Ewa; Dunin-Horkawicz, Stanislaw; Łasica, Anna M; Wosten, M.M.S.M; Nagy-Staroń, Anna; Godlewska, Renata; Bocian-Ostrzycka, Katarzyna; Pieńkowska, Katarzyna; Łaniewski, Paweł; Bujnicki, Janusz M; van Putten, Jos P M; Jagusztyn-Krynicka, E Katarzyna

    2014-01-01

    BACKGROUND: Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the funct

  6. INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.

    Science.gov (United States)

    Morrison, Ashby J; Highland, Jessica; Krogan, Nevan J; Arbel-Eden, Ayelet; Greenblatt, Jack F; Haber, James E; Shen, Xuetong

    2004-12-17

    While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

  7. Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

    Science.gov (United States)

    Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

    2014-04-01

    Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

  8. Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Zhiming [Department of Neurosurgery, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan (China); Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Wang Ping; Wang Hongyan; Zhang Xiangming [Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Wang Minli [Division of Life Sciences, Universities Space Research Association, Houston, Texas (United States); Cucinotta, Francis A. [National Aeronautics and Space Administration, Lyndon B. Johnson Space Center, Houston, Texas (United States); Wang Ya, E-mail: ywang94@emory.edu [Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)

    2013-02-01

    Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials: Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.

  9. Increasing the efficiency of homologous recombination vec-tor-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts%siRNA 干扰绵羊胚胎成纤维细胞 Lig4基因增加同源重组载体重连修复效率

    Institute of Scientific and Technical Information of China (English)

    王伟; 王玉霜; 黄兰兰; 简子健; 王新华; 刘守仁; 皮文辉

    2016-01-01

    In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to ef-fectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene pro-vides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts.%在动物细胞中,抑制非同源末端连接(Non-homologous end joining, NHEJ)修复途径,可以提高同源重组(Homologous recombination, HR)修复基因组双链断裂(Double-strand brakes, DSBs)的发生概率。为了提高绵羊胚胎成纤维细胞的 HR 效率,针对 NHEJ 修复途径中的关键因子 Lig4(DNA ligase 4)基因,本文设计合成4个具有靶向性的 siRNA(Small interfering RNA)。绵羊胚胎成纤维细胞经电转染导入 siRNA,通过实时荧光定量PCR(qRT-PCR)和 Western blotting 检测,筛选出有效抑制 Lig4基因表达的2个 siRNA。应用质粒重连法检测HR 修复效率,将 I-SceⅠ酶线性化的 HR 质粒和 siRNA 共转染绵羊胚胎成纤维细胞,经72 h 培养及流式细胞仪检测,与对照

  10. Dual-reporter surrogate systems for efficient enrichment of genetically modified cells.

    Science.gov (United States)

    Ren, Chonghua; Xu, Kun; Liu, Zhongtian; Shen, Juncen; Han, Furong; Chen, Zhilong; Zhang, Zhiying

    2015-07-01

    Isolation of genetically modified cells generated by designed nucleases are challenging, since they are often phenotypically indistinguishable from their parental cells. To efficiently enrich genetically modified cells, we developed two dual-reporter surrogate systems, namely NHEJ-RPG and SSA-RPG based on NHEJ and SSA repair mechanisms, respectively. Repair and enrichment efficiencies of these two systems were compared using different nucleases. In both CRISPR-Cas9- and ZFNs-induced DSB repair studies, we found that the efficiency and sensitivity of the SSA-RPG reporter with direct repeat length more than 200 bp were much higher than the NHEJ-RPG reporter. By utilizing the SSA-RPG reporter, we achieved the enrichment for indels in several endogenous loci with 6.3- to 34.8-fold of non-selected cells. Thus, the highly sensitive SSA-RPG reporter can be used for activity validation of designed nucleases and efficient enrichment of genetically modified cells. Besides, our systems offer alternative enrichment choices either by puromycin selection or FACS.

  11. Sirtuin 6 (SIRT6) rescues the decline of homologous recombination repair during replicative senescence

    Science.gov (United States)

    Mao, Zhiyong; Tian, Xiao; Van Meter, Michael; Ke, Zhonghe; Gorbunova, Vera; Seluanov, Andrei

    2012-01-01

    Genomic instability is a hallmark of aging tissues. Genomic instability may arise from the inefficient or aberrant function of DNA double-stranded break (DSB) repair. DSBs are repaired by homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). HR is a precise pathway, whereas NHEJ frequently leads to deletions or insertions at the repair site. Here, we used normal human fibroblasts with a chromosomally integrated HR reporter cassette to examine the changes in HR efficiency as cells progress to replicative senescence. We show that HR declines sharply with increasing replicative age, with an up to 38-fold decrease in efficiency in presenescent cells relative to young cells. This decline is not explained by a reduction of the number of cells in S/G2/M stage as presenescent cells are actively dividing. Expression of proteins involved in HR such as Rad51, Rad51C, Rad52, NBS1, and Sirtuin 6 (SIRT6) diminished with cellular senescence. Supplementation of Rad51, Rad51C, Rad52, and NBS1 proteins, either individually or in combination, did not rescue the senescence-related decline of HR. However, overexpression of SIRT6 in “middle-aged” and presenescent cells strongly stimulated HR repair, and this effect was dependent on mono-ADP ribosylation activity of poly(ADP-ribose) polymerase (PARP1). These results suggest that in aging cells, the precise HR pathway becomes repressed giving way to a more error-prone NHEJ pathway. These changes in the processing of DSBs may contribute to age-related genomic instability and a higher incidence of cancer with age. SIRT6 activation provides a potential therapeutic strategy to prevent the decline in genome maintenance. PMID:22753495

  12. Functional Analysis of Homologous Recombination Repair Proteins HerA and NurA in the Thermophile Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Huang, Qihong

    A number of DNA lesions are generated in each cell every day, among which double-stranded breaks (DSBs) constitute one of the most detrimental types of DNA damage. DSBs lead to genome instability, cell death, or even tumorigenesis in human, if not repaired timely. Two main pathways are known...... for DSB repair, homologous recombination repair (HRR) and Non-homologous end joint (NHEJ). HR repairs DSBs using a homologous DNA molecule as a template resulting in error free DNA repair, whereas NHEJ promotes direct re-ligation of the broken DNA ends in an error-prone manner. In eukaryotes DSBs occurred...

  13. Effect of 2-deoxy-D-glucose on DNA double strand break repair, cell survival and energy metabolism in euoxic Ehrlich ascites tumour cells

    Energy Technology Data Exchange (ETDEWEB)

    Jha, B.; Pohlit, W. (L.N. Mithila Univ., Darbhanga (India). Botany Dept.)

    1992-10-01

    Effects of 2-deoxy-D-glucose (2-DG) on DNA double strand break (dsb) repair, cell survival and on the energy metabolism were investigated in exponentially growing Ehrlich ascites tumour (EAT) cells. Cells in suspension were exposed to 40 Gy of X-rays and allowed to repair (up to 4h) with or without 2-DG at 37[sup o]C. DNA dsb rejoining was measured by means of clamped homogeneous electric field (CHEF), a pulsed field gel electrophoresis technique. The fraction of activity released (FAR) during electrophoresis (DNA associated [sup 14]C-thymidine) was used as a parameter to determine the number of dsb present in the DNA. Biphasic kinetics for dsb repair were observed. The presence of 2-DG significantly inhibited the slow component of dsb repair. The presence of 2-DG also enhanced radiation-induced cell killing. ATP content of cells was measured by a bioluminescence method. ATP content in exponentially growing cells was about 4 pg per cell. The level of ATP was reduced by 50% in presence of 2-DG (C[sub 2-DG]/C[sub G] = 1.0). (author).

  14. The AtRAD21.1 and AtRAD21.3 Arabidopsis cohesins play a synergistic role in somatic DNA double strand break damage repair

    OpenAIRE

    2014-01-01

    Background The RAD21 cohesin plays, besides its well-recognised role in chromatid cohesion, a role in DNA double strand break (dsb) repair. In Arabidopsis there are three RAD21 paralog genes (AtRAD21.1, AtRAD21.2 and AtRAD21.3), yet only AtRAD21.1 has been shown to be required for DNA dsb damage repair. Further investigation of the role of cohesins in DNA dsb repair was carried out and is here reported. Results We show for the first time that not only AtRAD21.1 but also AtRAD21.3 play a role ...

  15. Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents.

    Science.gov (United States)

    Smith, Roxanne P; Paxman, Jason J; Scanlon, Martin J; Heras, Begoña

    2016-07-16

    Recent years have witnessed a dramatic increase in bacterial antimicrobial resistance and a decline in the development of novel antibiotics. New therapeutic strategies are urgently needed to combat the growing threat posed by multidrug resistant bacterial infections. The Dsb disulfide bond forming pathways are potential targets for the development of antimicrobial agents because they play a central role in bacterial pathogenesis. In particular, the DsbA/DsbB system catalyses disulfide bond formation in a wide array of virulence factors, which are essential for many pathogens to establish infections and cause disease. These redox enzymes are well placed as antimicrobial targets because they are taxonomically widespread, share low sequence identity with human proteins, and many years of basic research have provided a deep molecular understanding of these systems in bacteria. In this review, we discuss disulfide bond catalytic pathways in bacteria and their significance in pathogenesis. We also review the use of different approaches to develop inhibitors against Dsb proteins as potential anti-virulence agents, including fragment-based drug discovery, high-throughput screening and other structure-based drug discovery methods.

  16. The Concerted Run on the DSB Bank: An Exploratory System Dynamics Approach

    NARCIS (Netherlands)

    Pruyt, E.; Hamarat, C.

    2010-01-01

    In this paper, an Exploratory System Dynamics model of a concerted run is first of all presented. The immediate cause for modelling a concerted bank run was the mediatised call for a run on the DSB bank. This Exploratory System Dynamics model was developed the morning of the call for the bank run, b

  17. Constructing Periodic Timetables using MIP - a case study from DSB S-train

    DEFF Research Database (Denmark)

    Nielsen, Morten N.; Hove, Bjørn; Clausen, Jens

    2006-01-01

    We describe a mathematical model to create operational timetable alternatives in DSB S-tog a/s. The model is a mixed integer program implemented in GAMS and solved by CPLEX. We investigate the impact of automatic merges of lines and perform scenario analysis for a subset of the parameters...

  18. The contribution of alu elements to mutagenic DNA double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Maria E Morales

    2015-03-01

    Full Text Available Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%, we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences. Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence

  19. Analysis of chromatin structure at meiotic DSB sites in yeasts.

    Science.gov (United States)

    Hirota, Kouji; Fukuda, Tomoyuki; Yamada, Takatomi; Ohta, Kunihiro

    2009-01-01

    One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion ofchromatin DNA and subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several-kb regions of interest and can be applied to other purposes.

  20. Reward optimization of a repairable system

    Energy Technology Data Exchange (ETDEWEB)

    Castro, I.T. [Departamento de Matematicas, Facultad de Veterinaria, Universidad de Extremadura, Avenida de la Universidad, s/n. 10071 Caceres (Spain)]. E-mail: inmatorres@unex.es; Perez-Ocon, R. [Departamento de Estadistica e Investigacion Operativa, Facultad de Ciencias, Universidad de Granada, Avenida de Severo Ochoa, s/n. 18071 Granada (Spain)]. E-mail: rperezo@ugr.es

    2006-03-15

    This paper analyzes a system subject to repairable and non-repairable failures. Non-repairable failures lead to replacement of the system. Repairable failures, first lead to repair but they lead to replacement after a fixed number of repairs. Operating and repair times follow phase type distributions (PH-distributions) and the pattern of the operating times is modelled by a geometric process. In this context, the problem is to find the optimal number of repairs, which maximizes the long-run average reward per unit time. To this end, the optimal number is determined and it is obtained by efficient numerical procedures.

  1. Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Martini

    2011-09-01

    Full Text Available Meiotic DNA double-strand breaks (DSBs initiate crossover (CO recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs. Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs. First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

  2. p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

    Science.gov (United States)

    Gong, Lu; Gong, Hongjian; Pan, Xiao; Chang, Changqing; Ou, Zhao; Ye, Shengfan; Yin, Le; Yang, Lina; Tao, Ting; Zhang, Zhenhai; Liu, Cong; Lane, David P; Peng, Jinrong; Chen, Jun

    2015-03-01

    The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

  3. Quantitative analysis reveals asynchronous and more than DSB-associated histone H2AX phosphorylation after exposure to ionizing radiation.

    Science.gov (United States)

    Han, Jianxun; Hendzel, Michael J; Allalunis-Turner, Joan

    2006-03-01

    Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called gamma-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual gamma-H2AX foci. In addition, there are hundreds of smaller gamma-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated gamma-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each gamma-H2AX focus as well as to discriminate DSB-related gamma-H2AX foci from DSB-unrelated gamma-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.

  4. Proteasome nuclear import mediated by Arc3 can influence efficient DNA damage repair and mitosis in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Cabrera, Rodrigo; Sha, Zhe; Vadakkan, Tegy J.;

    2010-01-01

    Proteasomes must remove regulatory molecules and abnormal proteins throughout the cell, but how proteasomes can do so efficiently remains unclear. We have isolated a subunit of the Arp2/3 complex, Arc3, which binds proteasomes. When overexpressed, Arc3 rescues phenotypes associated with proteasom...

  5. Proteasome nuclear import mediated by Arc3 can influence efficient DNA damage repair and mitosis in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Cabrera, Rodrigo; Sha, Zhe; Vadakkan, Tegy J.

    2010-01-01

    . Proteasome nuclear import is reduced when Arc3 is inactivated, leading to hypersensitivity to DNA damage and inefficient cyclin-B degradation, two events occurring in the nucleus. These data suggest that proteasomes display Arc3-dependent mobility in the cell, and mobile proteasomes can efficiently access...

  6. DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

    Energy Technology Data Exchange (ETDEWEB)

    Urushihara, Yusuke [Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562 (Japan); Kobayashi, Junya [Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto 606-8501 (Japan); Matsumoto, Yoshihisa [Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550 (Japan); Komatsu, Kenshi [Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto 606-8501 (Japan); Oda, Shoji [Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562 (Japan); Mitani, Hiroshi, E-mail: mitani@k.u-tokyo.ac.jp [Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562 (Japan)

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. Black-Right-Pointing-Pointer A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. Black-Right-Pointing-Pointer DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. Black-Right-Pointing-Pointer DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. Black-Right-Pointing-Pointer DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after {gamma}-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of {gamma}H2AX foci after {gamma}-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of {gamma}H2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after {gamma}-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the

  7. Regulation of ATM in DNA double strand break repair accounts for the radiosensitivity in human cells exposed to high linear energy transfer ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Xue Lian, E-mail: xuelian@suda.edu.cn [School of Radiation Medicine and Public Health, Medical College of Soochow University, No. 199, Ren' ai Road, Suzhou 215123 (China); Yu Dong, E-mail: ydong@ncc.go.jp [Tumor Endocrinology Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Furusawa, Yoshiya; Okayasu, Ryuichi [Heavy-Ion Radiobiology Research Group, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi 263-8555 (Japan); Tong Jian; Cao Jianping; Fan Saijun [School of Radiation Medicine and Public Health, Medical College of Soochow University, No. 199, Ren' ai Road, Suzhou 215123 (China)

    2009-11-02

    High linear energy transfer (LET) radiation shows different biological effects from low-LET radiation. The complex nature of high LET radiation-induced damage, especially the clustered DNA damage, brings about slow repair of DNA double strand breaks (DSBs), which finally lead to higher lethality and chromosome aberration. Ionizing radiation (IR) induced DNA DSBs are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) pathways in mammalian cells. The novel function of ataxia telangiectasia-mutated (ATM) protein is its involvement in the DSB repair of slow kinetics for 'dirty' breaks rejoining by NHEJ, this suggests that ATM may play a more important role in high LET radiation-induced DNA damage. We show here that KU55933, an ATM inhibitor could distinctly lower the clonogenic survival in normal human skin fibroblast cells exposed to carbon ion radiation and dramatically impair the normal process for DSB repair. We also implicated the involvement of ATM in the two pathways of DNA DSB repair, with DNA-PKcs and Rad51 as the representative proteins. The phosphorylation of DNA-PKcs at Thr-2609 with both immunoblotting and immunofluorescent staining indicated an ATM-dependent change, while for Rad51, KU55933 pretreatment could postpone the formation of nuclear Rad51 foci. Interestingly, we also found that pretreatment with chloroquine, an ATM stimulator could protect cells from carbon ion radiation only at lower doses. For doses over 1 Gy, protection was no longer observed. There was a dose-dependent increase for ATM kinase activity, with saturation at about 1 Gy. Chloroquine pretreatment prior to 1 Gy of carbon ion radiation did not enhance the autophosphorylation of ATM at serine 1981. The function of ATM in G2/M checkpoint arrest facilitated DSB repair in high-LET irradiation. Our results provide a possible mechanism for the direct involvement of ATM in DSB repair by high-LET irradiation.

  8. Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness

    DEFF Research Database (Denmark)

    Kari, Vijayalakshmi; Mansour, Wael Yassin; Raul, Sanjay Kumar

    2016-01-01

    IP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR-mediated DNA repair but not non......-homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may......The CHD1 gene, encoding the chromo-domain helicase DNA-binding protein-1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double-strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of Ct...

  9. DNA double-strand break repair: a tale of pathway choices

    Institute of Scientific and Technical Information of China (English)

    Jing Li; Xingzhi Xu

    2016-01-01

    Deoxyribonucleic acid double-strand breaks (DSBs) are cytotoxic lesions that must be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways.DSB repair is critical for genome integrity,cellular homeostasis and also constitutes the biological foundation for radiotherapy and the majority of chemotherapy.The choice between HR and NHEJ is a complex yet not completely understood process that will entail more future efforts.Herein we review our current understandings about how the choice is made over an antagonizing balance between p53-binding protein 1 and breast cancer 1 in the context of cell cycle stages,downstream effects,and distinct chromosomal histone marks.These exciting areas of research will surely bring more mechanistic insights about DSB repair and be utilized in the clinical settings.

  10. Repairs of composite structures

    Science.gov (United States)

    Roh, Hee Seok

    Repair on damaged composite panels was conducted. To better understand adhesively bonded repair, the study investigates the effect of design parameters on the joint strength. The design parameters include bondline length, thickness of adherend and type of adhesive. Adhesives considered in this study were tested to measure their tensile material properties. Three types of adhesively bonded joints, single strap, double strap, and single lap joint were considered under changing bondline lengths, thickness of adherend and type of adhesive. Based on lessons learned from bonded joints, a one-sided patch repair method for composite structures was conducted. The composite patch was bonded to the damaged panel by either film adhesive FM-73M or paste adhesive EA-9394 and the residual strengths of the repaired specimens were compared under varying patch sizes. A new repair method using attachments has been suggested to enhance the residual strength. Results obtained through experiments were analyzed using finite element analysis to provide a better repair design and explain the experimental results. It was observed that the residual strength of the repaired specimen was affected by patch length. Method for rapid repairs of damaged composite structures was investigated. The damage was represented by a circular hole in a composite laminated plate. Pre-cured composite patches were bonded with a quick-curing commercial adhesive near (rather than over) the hole. Tensile tests were conducted on specimens repaired with various patch geometries. The test results showed that, among the methods investigated, the best repair method restored over 90% of the original strength of an undamaged panel. The interfacial stresses in the adhesive zone for different patches were calculated in order to understand the efficiencies of the designs of these patch repairs. It was found that the composite patch that yielded the best strength had the lowest interfacial peel stress between the patch and

  11. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  12. MOF Phosphorylation by ATM Regulates 53BP1-Mediated Double-Strand Break Repair Pathway Choice

    Directory of Open Access Journals (Sweden)

    Arun Gupta

    2014-07-01

    Full Text Available Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ or homologous recombination (HR. Here, we report that double-strand breaks (DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase phosphorylation (p-T392-MOF and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  13. CtIP is required to initiate replication-dependent interstrand crosslink repair.

    Directory of Open Access Journals (Sweden)

    Michelle L Duquette

    Full Text Available DNA interstrand crosslinks (ICLs are toxic lesions that block the progression of replication and transcription. CtIP is a conserved DNA repair protein that facilitates DNA end resection in the double-strand break (DSB repair pathway. Here we show that CtIP plays a critical role during initiation of ICL processing in replicating human cells that is distinct from its role in DSB repair. CtIP depletion sensitizes human cells to ICL inducing agents and significantly impairs the accumulation of DNA damage response proteins RPA, ATR, FANCD2, γH2AX, and phosphorylated ATM at sites of laser generated ICLs. In contrast, the appearance of γH2AX and phosphorylated ATM at sites of laser generated double strand breaks (DSBs is CtIP-independent. We present a model in which CtIP functions early in ICL repair in a BRCA1- and FANCM-dependent manner prior to generation of DSB repair intermediates.

  14. Tendon repair

    Science.gov (United States)

    Repair of tendon ... Tendon repair can be performed using: Local anesthesia (the immediate area of the surgery is pain-free) ... a cut on the skin over the injured tendon. The damaged or torn ends of the tendon ...

  15. Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes.

    Science.gov (United States)

    Zhou, Xiaoyang; Wang, Lulu; Du, Yinan; Xie, Fei; Li, Liang; Liu, Yu; Liu, Chuanhong; Wang, Shiqiang; Zhang, Shibing; Huang, Xingxu; Wang, Yong; Wei, Hong

    2016-01-01

    Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced homology-directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non-homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR-derived mutation, in this work, without inhibition of NHEJ, we first generated gene-modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9-induced HDR in zygotes using single-strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9-induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR-derived mutation in pig zygotes, suggesting a possible balance for optimal HDR-derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR-derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off-targeting, suggesting CRISPR/Cas9-induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs.

  16. Deregulation of DNA double-strand break repair in multiple myeloma: implications for genome stability.

    Directory of Open Access Journals (Sweden)

    Ana B Herrero

    Full Text Available Multiple myeloma (MM is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since both impaired and hyperactive double strand break (DSB repair pathways can result in DNA rearrangements, we investigated the functionality of DSB repair in MM cells. Repair kinetics of ionizing-radiation (IR-induced DSBs was similar in MM and normal control lymphoblastoid cell lines, as revealed by the comet assay. However, four out of seven MM cell lines analyzed exhibited a subset of persistent DSBs, marked by γ-H2AX and Rad51 foci that elicited a prolonged G2/M DNA damage checkpoint activation and hypersensitivity to IR, especially in the presence of checkpoint inhibitors. An analysis of the proteins involved in DSB repair in MM cells revealed upregulation of DNA-PKcs, Artemis and XRCC4, that participate in non-homologous end joining (NHEJ, and Rad51, involved in homologous recombination (HR. Accordingly, activity of both NHEJ and HR were elevated in MM cells compared to controls, as determined by in vivo functional assays. Interestingly, levels of proteins involved in a highly mutagenic, translocation-promoting, alternative NHEJ subpathway (Alt-NHEJ were also increased in all MM cell lines, with the Alt-NHEJ protein DNA ligase IIIα, also overexpressed in several plasma cell samples isolated from MM patients. Overactivation of the Alt-NHEJ pathway was revealed in MM cells by larger deletions and higher sequence microhomology at repair junctions, which were reduced by chemical inhibition of the pathway. Taken together, our results uncover a deregulated DSB repair in MM that might underlie the characteristic genome instability of the disease, and could be therapeutically exploited.

  17. Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria.

    Science.gov (United States)

    Um, Si-Hyeon; Kim, Jin-Sik; Song, Saemee; Kim, Nam Ah; Jeong, Seong Hoon; Ha, Nam-Chul

    2015-08-01

    In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

  18. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  19. DNA Repair Defects and Chromosomal Aberrations

    Science.gov (United States)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  20. 基于SystemView的DSB-SC通信系统的仿真与分析

    Institute of Scientific and Technical Information of China (English)

    谢莉

    2015-01-01

    SystemView是一种适于通信系统设计与仿真分析的软件工具.可以对通信系统的工作过程进行实时仿真分析,文章以DSB-SC通信系统为例,利用SystemView仿真软件对DSB-SC的调制解调过程进行了仿真.仿真结果表明,利用SystemView仿真软件可以很方便地得到通信系统的输出结果,从而说明电路各参数对系统性能的影响.

  1. Rolling Stock Planning at DSB S-tog - Processes, Cost Structures and Requirements

    DEFF Research Database (Denmark)

    Thorlacius, Per

    A central issue for operators of suburban passenger train transport systems is providing sufficient number of seats for the passengers while at the same time minimising operating costs. The process of providing this is called rolling stock planning. This technical report documents the terminology......, the processes, the cost structures and the requirements for rolling stock planning at DSB S-tog, the suburban passenger train operator of the City of Copenhagen. The focus of the technical report is directed at practical train operator oriented issues. The technical report is thought to serve as a basis...... for investigating better methods to perform the rolling stock planning (to be the topic of later papers). This technical report is produced as a part of the current industrial Ph. D. project to improve the rolling stock planning process of DSB S-tog....

  2. Synthetic lethal targeting of DNA double strand break repair deficient cells by human apurinic/apyrimidinic endonuclease (APE1) inhibitors

    OpenAIRE

    Sultana, Rebeka; McNeill, Daniel R.; Abbotts, Rachel; Mohammed, Mohammed Z.; Zdzienicka, Małgorzata Z.; Qutob, Haitham; Seedhouse, Claire; Charles A. Laughton; Fischer, Peter M.; Patel, Poulam M.; Wilson, David M.; Madhusudan, Srinivasan

    2012-01-01

    An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In the current study we have investigated the ability of APE1 inhibitors to induce synthetic lethality in a panel of DNA double strand break (DSB) repair deficient and proficient cells; a) Chine...

  3. Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex.

    Science.gov (United States)

    Dutta, Arijit; Eckelmann, Bradley; Adhikari, Sanjay; Ahmed, Kazi Mokim; Sengupta, Shiladitya; Pandey, Arvind; Hegde, Pavana M; Tsai, Miaw-Sheue; Tainer, John A; Weinfeld, Michael; Hegde, Muralidhar L; Mitra, Sankar

    2017-03-17

    Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. MRE11 is required for homologous synapsis and DSB processing in rice meiosis.

    Science.gov (United States)

    Ji, Jianhui; Tang, Ding; Wang, Mo; Li, Yafei; Zhang, Lei; Wang, Kejian; Li, Ming; Cheng, Zhukuan

    2013-10-01

    Mre11, a conserved protein found in organisms ranging from yeast to multicellular organisms, is required for normal meiotic recombination. Mre11 interacts with Rad50 and Nbs1/Xrs2 to form a complex (MRN/X) that participates in double-strand break (DSB) ends processing. In this study, we silenced the MRE11 gene in rice and detailed its function using molecular and cytological methods. The OsMRE11-deficient plants exhibited normal vegetative growth but could not set seed. Cytological analysis indicated that in the OsMRE11-deficient plants, homologous pairing was totally inhibited, and the chromosomes were completely entangled as a formation of multivalents at metaphase I, leading to the consequence of serious chromosome fragmentation during anaphase I. Immunofluorescence studies further demonstrated that OsMRE11 is required for homologous synapsis and DSB processing but is dispensable for meiotic DSB formation. We found that OsMRE11 protein was located on meiotic chromosomes from interphase to late pachytene. This protein showed normal localization in zep1, Oscom1 and Osmer3, as well as in OsSPO11-1(RNAi) plants, but not in pair2 and pair3 mutants. Taken together, our results provide evidence that OsMRE11 performs a function essential for maintaining the normal HR process and inhibiting non-homologous recombination during meiosis.

  5. High atomic weight, high-energy radiation (HZE induces transcriptional responses shared with conventional stresses in addition to a core DSB response specific to clastogenic treatments.

    Directory of Open Access Journals (Sweden)

    Victor eMissirian

    2014-08-01

    Full Text Available Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as collateral damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR treatment, HZE (1 GeV Fe26+ high mass, high charge, and high energy relativistic particles and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs, but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5 to 24 hours after irradiation in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the DSB response were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 hours post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 hr after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing extended night. This response was not apparent in gamma-irradiated plants.

  6. High atomic weight, high-energy radiation (HZE) induces transcriptional responses shared with conventional stresses in addition to a core "DSB" response specific to clastogenic treatments.

    Science.gov (United States)

    Missirian, Victor; Conklin, Phillip A; Culligan, Kevin M; Huefner, Neil D; Britt, Anne B

    2014-01-01

    Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as "collateral" damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe(26+) high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the "DSB response" were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing "extended night." This response was not apparent in gamma-irradiated plants.

  7. Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism

    Directory of Open Access Journals (Sweden)

    Grabowska Anna D

    2011-07-01

    Full Text Available Abstract Background Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in Campylobacter jejuni is more complex than the one in the laboratory E. coli K-12 strain. Results In the C. jejuni 81-176 genome, the dsb genes of the oxidative pathway are arranged in three transcriptional units: dsbA2-dsbB-astA, dsbA1 and dba-dsbI. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in dsb gene regulation was proven by a reporter gene study in a C. jejuni wild type strain and its isogenic fur mutant. An electrophoretic mobility shift assay (EMSA confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind in vitro to the C. jejuni promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives dsbA1 gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary dba-dsbI mRNA structure for dsbI mRNA translation was verified by estimating individual dsbI gene expression from its own promoter. Conclusions The present work shows that iron concentration is a significant factor in dsb gene transcription. These results support the concept that iron concentration - also through its influence on dsb gene expression - might control the abundance of extracytoplasmic proteins

  8. Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

    Science.gov (United States)

    Cohen, Shahar; Leshansky, Lucy; Zussman, Eyal; Burman, Michael; Srouji, Samer; Livne, Erella; Abramov, Natalie; Itskovitz-Eldor, Joseph

    2010-10-01

    The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

  9. Bladder exstrophy repair

    Science.gov (United States)

    Bladder birth defect repair; Everted bladder repair; Exposed bladder repair; Repair of bladder exstrophy ... Bladder exstrophy repair involves two surgeries. The first surgery is to repair the bladder and the second one is to attach ...

  10. The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions

    NARCIS (Netherlands)

    Moser, J; Volker, M; Kool, H; Alekseev, S; Vrieling, H; Yasui, A; van Zeeland, AA; Mullenders, LHF

    2005-01-01

    Previous studies point to the XPC-hHR23B complex as the principal initiator of global genome nucleotide excision repair (NER) pathway, responsible for the repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) in human cells. However, the UV-damaged DNA binding protei

  11. An Approach to Elucidate NBS1 Function in DNA Repair Using Frequent Nonsynonymous Polymorphism in Wild Medaka (Oryzias latipes) Populations.

    Science.gov (United States)

    Igarashi, Kento; Kobayashi, Junya; Katsumura, Takafumi; Urushihara, Yusuke; Hida, Kyohei; Watanabe-Asaka, Tomomi; Oota, Hiroki; Oda, Shoji; Mitani, Hiroshi

    2017-01-01

    Nbs1 is one of the genes responsible for Nijmegen breakage syndrome, which is marked with high radiosensitivity. In human NBS1 (hNBS1), Q185E polymorphism is known as the factor to cancer risks, although its DSB repair defect has not been addressed. Here we investigated the genetic variations in medaka (Oryzias latipes) wild populations, and found 40 nonsynonymous single nucleotide polymorphisms (SNPs) in medaka nbs1 (olnbs1) gene within 5 inbred strains. A mutation to histidine in Q170 residue in olNbs1, which corresponds to Q185 residue of hNBS1, was widely distributed in the closed colonies derived from the eastern Korean population of medaka. Overexpression of H170 type olNbs1 in medaka cultured cell lines resulted in the increased accumulation of olNbs1 at laser-induced DSB sites. Autophosphorylation of DNA-dependent protein kinase at T2609 was suppressed after the γ-ray irradiation, which was followed by prolonged formation of γ-H2AX foci and delayed DSB repair. These findings suggested that the nonsynonymous SNP (Q170H) in olnbs1, which induced DSB repair defects, is specifically distributed in the eastern Korean population of medaka. Furthermore, examination using the variation within wild populations might provide a novel method to characterize a driving force to spread the disease risk alleles.

  12. Impact of two DNA repair pathways, homologous recombination and non-homologous end joining, on bacterial spore inactivation under simulated martian environmental conditions

    Science.gov (United States)

    Moeller, Ralf; Schuerger, Andrew C.; Reitz, Günther; Nicholson, Wayne L.

    2011-09-01

    Spores of Bacillus subtilis were used as a model system to study the impact of the two major DNA double-strand break (DSB) repair mechanisms [homologous recombination (HR) and non-homologous end-joining (NHEJ)] on the survivability of air-dried mono- and multilayers of bacterial spores under a simulated martian environment; i.e., an environment with low temperature (-10 °C), pure CO 2 atmosphere (99.99% CO 2), 200-1100 nm UV-VIS-NIR radiation, and 0.69 kPa pressure. Spores in multilayers exhibited low inactivation rates compared to monolayers, mainly due to shadowing effects of overlying spores. Simulated martian UV irradiation reduced dramatically spore viability, whereas when shielded from martian UV radiation, spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to simulated martian environmental conditions than were wild-type spores. In addition, NHEJ-deficient spores were consistently more sensitive than HR-deficient spores to simulated Mars environmental conditions, suggesting that DSBs were an important type of DNA damage. The results indicated that both HR and NHEJ provide an efficient set of DNA repair pathways ensuring spore survival after exposure to simulated martian environmental conditions.

  13. Modulation of biofilm-formation in Salmonella enterica serovar Typhimurium by the periplasmic DsbA/DsbB oxidoreductase system requires the GGDEF-EAL domain protein STM3615.

    Directory of Open Access Journals (Sweden)

    Naeem Anwar

    Full Text Available In Salmonella enterica serovar Typhimurium (S. Typhimurium, biofilm-formation is controlled by the cytoplasmic intracellular small-molecular second messenger cyclic 3', 5'-di- guanosine monophosphate (c-di-GMP through the activities of GGDEF and EAL domain proteins. Here we describe that deleting either dsbA or dsbB, respectively encoding a periplasmic protein disulfide oxidase and a cytoplasmic membrane disulfide oxidoreductase, resulted in increased biofilm-formation on solid medium. This increased biofilm-formation, defined as a red, dry and rough (rdar colony morphotype, paralleled with enhanced expression of the biofilm master regulator CsgD and the biofilm-associated fimbrial subunit CsgA. Deleting csgD in either dsb mutant abrogated the enhanced biofilm-formation. Likewise, overexpression of the c-di-GMP phosphodiesterase YhjH, or mutationally inactivating the CsgD activator EAL-domain protein YdiV, reduced biofilm-formation in either of the dsb mutants. Intriguingly, deleting the GGDEF-EAL domain protein gene STM3615 (yhjK, previously not connected to rdar morphotype development, also abrogated the escalated rdar morphotype formation in dsb mutant backgrounds. Enhanced biofilm-formation in dsb mutants was furthermore annulled by exposure to the protein disulfide catalyst copper chloride. When analyzed for the effect of exogenous reducing stress on biofilm-formation, both dsb mutants initially showed an escalated rdar morphotype development that later dissolved to reveal a smooth mucoid colony morphotype. From these results we conclude that biofilm-development in S. Typhimurium is affected by periplasmic protein disulphide bond status through CsgD, and discuss the involvement of selected GGDEF/EAL domain protein(s as signaling mediators.

  14. BioSentinel: Mission Development of a Radiation Biosensor to Gauge DNA Damage and Repair Beyond Low Earth Orbit on a 6U Nanosatellite

    Science.gov (United States)

    Sanchez, Hugo; Lewis, Brian; Hanel, Robert

    2015-01-01

    We are designing and developing a 6U (10 x 22 x 34 cm; 14 kg) nanosatellite as a secondary payload to fly aboard NASAs Space Launch System (SLS) Exploration Mission (EM) 1, scheduled for launch in late 2017. For the first time in over forty years, direct experimental data from biological studies beyond low Earth orbit (LEO) will be obtained during BioSentinels 12- to 18-month mission. BioSentinel will measure the damage and repair of DNA in a biological organism and allow us to compare that to information from onboard physical radiation sensors. In order to understand the relative contributions of the space environments two dominant biological perturbations, reduced gravity and ionizing radiation, results from deep space will be directly compared to data obtained in LEO (on ISS) and on Earth. These data points will be available for validation of existing biological radiation damage and repair models, and for extrapolation to humans, to assist in mitigating risks during future long-term exploration missions beyond LEO. The BioSentinel Payload occupies 4U of the spacecraft and will utilize the monocellular eukaryotic organism Saccharomyces cerevisiae (yeast) to report DNA double-strand-break (DSB) events that result from ambient space radiation. DSB repair exhibits striking conservation of repair proteins from yeast to humans. Yeast was selected because of 1) its similarity to cells in higher organisms, 2) the well-established history of strains engineered to measure DSB repair, 3) its spaceflight heritage, and 4) the wealth of available ground and flight reference data. The S. cerevisiae flight strain will include engineered genetic defects to prevent growth and division until a radiation-induced DSB activates the yeasts DNA repair mechanisms. The triggered culture growth and metabolic activity directly indicate a DSB and its successful repair. The yeast will be carried in the dry state within the 1-atm PL container in 18 separate fluidics cards with each card

  15. Excess single-stranded DNA inhibits meiotic double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Rebecca Johnson

    2007-11-01

    Full Text Available During meiosis, self-inflicted DNA double-strand breaks (DSBs are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE, in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects

  16. Efficiency

    NARCIS (Netherlands)

    I.P. van Staveren (Irene)

    2009-01-01

    textabstractThe dominant economic theory, neoclassical economics, employs a single economic evaluative criterion: efficiency. Moreover, it assigns this criterion a very specific meaning. Other – heterodox – schools of thought in economics tend to use more open concepts of efficiency, related to comm

  17. PARP-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis?

    Energy Technology Data Exchange (ETDEWEB)

    Swindall, Amanda F.; Stanley, Jennifer A. [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Yang, Eddy S., E-mail: eyang@uab.edu [Department of Radiation Oncology Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, 176F HSROC Suite 2232B, 1700 6th Avenue South, Birmingham, AL 35249 (United States); Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States); Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35249 (United States)

    2013-07-26

    Oxidative stress induced by reactive oxygen species can result in DNA damage within cells and subsequently increase risk for carcinogenesis. This may be averted by repair of DNA damage through the base or nucleotide excision repair (BER/NER) pathways. PARP, a BER protein, is known for its role in DNA-repair. However, multiple lesions can occur within a small range of DNA, known as oxidative clustered DNA lesions (OCDLs), which are difficult to repair and may lead to the more severe DNA double-strand break (DSB). Inefficient DSB repair can then result in increased mutagenesis and neoplastic transformation. OCDLs occur more frequently within a variety of tumor tissues. Interestingly, PARP is highly expressed in several human cancers. Additionally, chronic inflammation may contribute to tumorigenesis through ROS-induced DNA damage. Furthermore, PARP can modulate inflammation through interaction with NFκB and regulating the expression of inflammatory signaling molecules. Thus, the upregulation of PARP may present a double-edged sword. PARP is needed to repair ROS-induced DNA lesions, but PARP expression may lead to increased inflammation via upregulation of NFκB signaling. Here, we discuss the role of PARP in the repair of oxidative damage versus the formation of OCDLs and speculate on the feasibility of PARP inhibition for the treatment and prevention of cancers by exploiting its role in inflammation.

  18. Depletion of the bloom syndrome helicase stimulates homology-dependent repair at double-strand breaks in human chromosomes.

    Science.gov (United States)

    Wang, Yibin; Smith, Krissy; Waldman, Barbara Criscuolo; Waldman, Alan S

    2011-04-03

    Mutation of BLM helicase causes Blooms syndrome, a disorder associated with genome instability, high levels of sister chromatid exchanges, and cancer predisposition. To study the influence of BLM on double-strand break (DSB) repair in human chromosomes, we stably transfected a normal human cell line with a DNA substrate that contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI. The substrate also contained a closely linked functional tk gene to serve as a recombination partner for the tk-neo fusion gene. We derived two cell lines each containing a single integrated copy of the DNA substrate. In these cell lines, a DSB was introduced within the tk-neo fusion gene by expression of I-SceI. DSB repair events that occurred via homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recovered by selection for G418-resistant clones. DSB repair was examined under conditions of either normal BLM expression or reduced BLM expression brought about by RNA interference. We report that BLM knockdown in both cell lines specifically increased the frequency of HR events that produced deletions by crossovers or single-strand annealing while leaving the frequency of gene conversions unchanged or reduced. We observed no change in the accuracy of individual HR events and no substantial alteration of the nature of individual NHEJ events when BLM expression was reduced. Our work provides the first direct evidence that BLM influences DSB repair pathway choice in human chromosomes and suggests that BLM deficiency can engender genomic instability by provoking an increased frequency of HR events of a potentially deleterious nature.

  19. A Role for BLM in Double-Strand Break Repair Pathway Choice: Prevention of CtIP/Mre11-Mediated Alternative Nonhomologous End-Joining

    DEFF Research Database (Denmark)

    Grabarz, Anastazja; Guirouilh-Barbat, Josée; Barascu, Aurelia

    2013-01-01

    The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200 bp) generated by alternative end-joining (A-EJ). BLM r...

  20. Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair

    NARCIS (Netherlands)

    Y.J. Choi (Yong Jun); H. Li (Han); M.Y. Son (Mi Young); X.-H. Wang (Xiao-Hong); J.L. Fornsaglio (Jamie L.); R.W. Sobol (Robert W.); M. Lee (Moonsook); J. Vijg (Jan); S. Imholz (Sandra); M.E.T. Dollé (Martijn); H. van Steeg (Harry); E. Reiling (Erwin); P. Hasty (Paul)

    2014-01-01

    textabstractKu70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB

  1. An in vitro DNA double-strand break repair assay based on end-joining of defined duplex oligonucleotides.

    Science.gov (United States)

    Datta, Kamal; Purkayastha, Shubhadeep; Neumann, Ronald D; Winters, Thomas A

    2012-01-01

    DNA double-strand breaks (DSBs) are caused by endogenous cellular processes such as oxidative metabolism, or by exogenous events like exposure to ionizing radiation or other genotoxic agents. Repair of these DSBs is essential for the maintenance of cellular genomic integrity. In human cells, and cells of other higher eukaryotes, DSBs are primarily repaired by the nonhomologous end-joining (NHEJ) DSB repair pathway. Most in vitro assays that have been designed to measure NHEJ activity employ linear plasmid DNA as end-joining substrates, and such assays have made significant contributions to our understanding of the biochemical mechanisms of NHEJ. Here we describe an in vitro end-joining assay employing linear oligonucleotides that has distinct advantages over plasmid-based assays for the study of structure-function relationships between the proteins of the NHEJ pathway and synthetic DNA end-joining substrates possessing predetermined DSB configurations and chemistries.

  2. Análisis funcional de DsbA una tiol oxidorreductasa de Rhizobium leguminosarum

    OpenAIRE

    Vega de los Reyes, Rosa Mª

    2009-01-01

    Las proteínas que se exportan fuera del citoplasma bacteriano constituyen hasta un 20% del total de proteínas sintetizadas. Algunas de estas proteínas necesitan adquirir su conformación tridimensional particular en el citoplasma, incorporando frecuentemente cofactores metálicos antes de su transporte. Dicho transporte está mediado por el sistema Tat que es capaz de transportar proteínas que presentan un péptido señal característico en conformación plegada. Se ha postulado DsbA como proteína d...

  3. Characterization of Helicobacter pylori HP0231 (DsbK): role in disulfide bond formation, redox homeostasis and production of Helicobacter cystein-rich protein HcpE.

    Science.gov (United States)

    Lester, Jeffrey; Kichler, Sari; Oickle, Brandon; Fairweather, Spencer; Oberc, Alexander; Chahal, Jaspreet; Ratnayake, Dinath; Creuzenet, Carole

    2015-04-01

    Helicobacter pylori is a human gastric pathogen that colonizes ∼ 50% of the world's population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. The numerous side effects of available treatments and the emergence of antibiotic resistant strains are severe concerns that justify further research into H. pylori's pathogenic mechanisms. H. pylori produces secreted proteins that may play a role in virulence, including the Helicobacter cysteine-rich protein HcpE (aka HP0235). We demonstrate herein that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles. We show that the structure of HcpE comprises an organized array of disulfide bonds. We identify DsbK (aka HP0231) as a folding factor necessary for HcpE production and secretion in H. pylori and show that recombinant DsbK can interact with and refold unprocessed, reduced HcpE in vitro. These experiments highlight the first biologically relevant substrate for DsbK. Furthermore, we show that DsbK has disulfide bond (Dsb) forming activity on reduced lysozyme and demonstrate a DsbA-type of activity for DsbK upon expression in E. coli, despite its similarity with DsbG. Finally, we show a role of DsbK in maintaining redox homeostasis in H. pylori.

  4. Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

    Directory of Open Access Journals (Sweden)

    Katarzyna Marta Bocian-Ostrzycka

    2015-10-01

    Full Text Available Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595 with a -propeller structure. The aim of presented work was to assess relations between HP0231 structure and function.We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C, a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in E. coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (Escherichia coli DsbA protein were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

  5. Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

    Science.gov (United States)

    Bocian-Ostrzycka, Katarzyna M; Łasica, Anna M; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J; Drabik, Karolina; Dobosz, Aneta M; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K

    2015-01-01

    Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

  6. In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA).

    Science.gov (United States)

    Wunderlich, M; Glockshuber, R

    1993-11-25

    The formation of disulfide bonds in Escherichia coli is catalyzed by periplasmic protein disulfide-isomerase (DsbA). When the alpha-amylase/trypsin inhibitor from Ragi, a protein containing five intramolecular disulfide bridges, is secreted into the periplasm of E. coli, large amounts of misfolded inhibitor with incomplete or incorrect disulfides are accumulated. Folding of the inhibitor in the periplasm is not improved when DsbA is coexpressed and cosecreted. However, an up to 14-fold increase in correctly folded inhibitor is observed by co-expression of DsbA in conjugation with the addition of reduced glutathione to the growth medium. This peptide acts as a disulfide-shuffling reagent and can pass the outer membrane of E. coli. Since the influence of DsbA on the folding yield of the inhibitor is reduced in the presence of oxidized glutathione, the in vivo function of DsbA appears to be dependent on the ratio between oxidizing and reducing thiol equivalents in the periplasm. The high stability of thiol reagents against air oxidation during growth of E. coli allows the investigation of oxidative protein folding in vivo under controlled, thiol-dependent redox conditions.

  7. The Arabidopsis thaliana DSB formation (AtDFO) gene is required for meiotic double-strand break formation.

    Science.gov (United States)

    Zhang, Cheng; Song, Yao; Cheng, Zhi-hao; Wang, Ying-xiang; Zhu, Jun; Ma, Hong; Xu, Ling; Yang, Zhong-Nan

    2012-10-01

    DNA double-strand break (DSB) formation is the initial event for meiotic recombination catalyzed by the conserved Spo11 protein. In Arabidopsis, several proteins have been reported to be involved in DSB formation. Here, we report an Arabidopsis DSB forming (DFO) gene in Arabidopsis that is involved in DSB formation. The dfo mutant exhibits reduced fertility, producing polyads with an abnormal number of microspores, unlike the tetrads in the wild type. The dfo meiocytes were defective in homologous chromosome synapsis and segregation. Genetic analysis revealed that the homologous recombination of Atdfo-1 is severely affected in meiotic prophase I. DFO encodes a protein without any known conserved domain. There was no homologue identified outside the plant kingdom, indicating that AtDFO is a plant-specific protein. AtMRE11 has been reported to be responsible for processing SPO11-generated DSBs. The Atmre11 mutant displays chromosome fragmentation during meiosis. However, the Atdfo Atmre11 double mutant had no such chromosome fragmentation, indicating that AtDFO is required for DSB formation.

  8. REPAIR MECHANISM IN FOREIGN LANGUAGE CLASSROOM INTERACTION

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This paper presents practical research on repair mechanismand its four repair trajectories in FL classroom interaction. Thisshows that it is effective and efficient in assisting FL learners todevelop their communicative competence and understand theprocess of language acquisition. Repair strategies that are ofgreat value to FL teachers in FL classroom teaching are also ex-pounded.

  9. High-Fidelity Reprogrammed Human IPSCs Have a High Efficacy of DNA Repair and Resemble hESCs in Their MYC Transcriptional Signature

    Directory of Open Access Journals (Sweden)

    Pratik K. Nagaria

    2016-01-01

    Full Text Available Human induced pluripotent stem cells (hiPSCs are reprogrammed from adult or progenitor somatic cells and must make substantial adaptations to ensure genomic stability in order to become “embryonic stem cell- (ESC- like.” The DNA damage response (DDR is critical for maintenance of such genomic integrity. Herein, we determined whether cell of origin and reprogramming method influence the DDR of hiPSCs. We demonstrate that hiPSCs derived from cord blood (CB myeloid progenitors (i.e., CB-iPSC via an efficient high-fidelity stromal-activated (sa method closely resembled hESCs in DNA repair gene expression signature and irradiation-induced DDR, relative to hiPSCs generated from CB or fibroblasts via standard methods. Furthermore, sa-CB-iPSCs also more closely resembled hESCs in accuracy of nonhomologous end joining (NHEJ, DNA double-strand break (DSB repair, and C-MYC transcriptional signatures, relative to standard hiPSCs. Our data suggests that hiPSCs derived via more efficient reprogramming methods possess more hESC-like activated MYC signatures and DDR signaling. Thus, an authentic MYC molecular signature may serve as an important biomarker in characterizing the genomic integrity in hiPSCs.

  10. P31comet, a member of the synaptonemal complex, participates in meiotic DSB formation in rice.

    Science.gov (United States)

    Ji, Jianhui; Tang, Ding; Shen, Yi; Xue, Zhihui; Wang, Hongjun; Shi, Wenqing; Zhang, Chao; Du, Guijie; Li, Yafei; Cheng, Zhukuan

    2016-09-20

    The human mitotic arrest-deficient 2 (Mad2) binding protein p31(comet) participates in the spindle checkpoint and coordinates cell cycle events in mitosis although its function in meiosis remains unknown in all organisms. Here, we reveal P31(comet) as a synaptonemal complex (SC) protein in rice (Oryza sativa L.). In p31(comet), homologous pairing and synapsis are eliminated, leading to the homologous nondisjunction and complete sterility. The failure in loading of histone H2AX phosphorylation (γH2AX) in p31(comet), together with the suppressed chromosome fragmentation in rice completion of meiotic recombination 1 (com1) p31(comet) and radiation sensitive 51c (rad51c) p31(comet) double mutants, indicates that P31(comet) plays an essential role in double-strand break (DSB) formation. Interestingly, the dynamic colocalization pattern between P31(comet) and ZEP1 (a transverse filament protein of SC) by immunostaining, as well as the interaction between P31(comet) and CENTRAL REGION COMPONENT 1 (CRC1) in yeast two-hybrid assays, suggests possible involvement of P31(comet) in SC installation. Together, these data indicate that P31(comet) plays a key role in DSB formation and SC installation, mainly through its cooperation with CRC1.

  11. Analytical model of chemical phase and formation of DSB in chromosomes by ionizing radiation.

    Science.gov (United States)

    Barilla, Jiří; Lokajíček, Miloš; Pisaková, Hana; Simr, Pavel

    2013-03-01

    Mathematical analytical model of the processes running in individual radical clusters during the chemical phase (under the presence of radiomodifiers) proposed by us earlier has been further developed and improved. It has been applied to the data presented by Blok and Loman characterizing the oxygen effect in SSB and DSB formation (in water solution and at low-LET radiation) also in the region of very small oxygen concentrations, which cannot be studied with the help of experiments done with living cells. In this new analysis the values of all reaction rates and diffusion parameters known from literature have been made use of. The great increase of SSB and DSB at zero oxygen concentration may follow from the fact that at small oxygen concentrations the oxygen absorbs other radicals while at higher concentrations the formation of oxygen radicals prevails. It explains the double oxygen effect found already earlier by Ewing. The model may be easily extended to include also the effects of other radiomodifiers present in medium during irradiation.

  12. Genetic variants in DNA double-strand break repair genes and risk of salivary gland carcinoma: a case-control study.

    Directory of Open Access Journals (Sweden)

    Li Xu

    Full Text Available DNA double strand break (DSB repair is the primary defense mechanism against ionizing radiation-induced DNA damage. Ionizing radiation is the only established risk factor for salivary gland carcinoma (SGC. We hypothesized that genetic variants in DSB repair genes contribute to individual variation in susceptibility to SGC. To test this hypothesis, we conducted a case-control study in which we analyzed 415 single nucleotide polymorphisms (SNPs in 45 DSB repair genes in 352 SGC cases and 598 controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs and 95% confidence intervals (CIs. Rs3748522 in RAD52 and rs13180356 in XRCC4 were significantly associated with SGC after Bonferroni adjustment; ORs (95% CIs for the variant alleles of these SNPs were 1.71 (1.40-2.09, P = 1.70 × 10(-7 and 0.58 (0.45-0.74, P = 2.00 × 10(-5 respectively. The genetic effects were modulated by histological subtype. The association of RAD52-rs3748522 with SGC was strongest for mucoepidermoid carcinoma (OR = 2.21, 95% CI: 1.55-3.15, P = 1.25 × 10(-5, n = 74, and the association of XRCC4-rs13180356 with SGC was strongest for adenoid cystic carcinoma (OR = 0.60, 95% CI: 0.42-0.87, P = 6.91 × 10(-3, n = 123. Gene-level association analysis revealed one gene, PRKDC, with a marginally significant association with SGC risk in non-Hispanic whites. To our knowledge, this study is the first to comprehensively evaluate the genetic effect of DSB repair genes on SGC risk. Our results indicate that genetic variants in the DSB repair pathways contribute to inter-individual differences in susceptibility to SGC and show that the impact of genetic variants differs by histological subtype. Independent studies are warranted to confirm these findings.

  13. Homologous recombination is a primary pathway to repair DNA double-strand breaks generated during DNA rereplication.

    Science.gov (United States)

    Truong, Lan N; Li, Yongjiang; Sun, Emily; Ang, Katrina; Hwang, Patty Yi-Hwa; Wu, Xiaohua

    2014-10-17

    Re-initiation of DNA replication at origins within a given cell cycle would result in DNA rereplication, which can lead to genome instability and tumorigenesis. DNA rereplication can be induced by loss of licensing control at cellular replication origins, or by viral protein-driven multiple rounds of replication initiation at viral origins. DNA double-strand breaks (DSBs) are generated during rereplication, but the mechanisms of how these DSBs are repaired to maintain genome stability and cell viability are poorly understood in mammalian cells. We generated novel EGFP-based DSB repair substrates, which specifically monitor the repair of rereplication-associated DSBs. We demonstrated that homologous recombination (HR) is an important mechanism to repair rereplication-associated DSBs, and sister chromatids are used as templates for such HR-mediated DSB repair. Micro-homology-mediated non-homologous end joining (MMEJ) can also be used but to a lesser extent compared to HR, whereas Ku-dependent classical non-homologous end joining (C-NHEJ) has a minimal role to repair rereplication-associated DSBs. In addition, loss of HR activity leads to severe cell death when rereplication is induced. Therefore, our studies identify HR, the most conservative repair pathway, as the primary mechanism to repair DSBs upon rereplication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs).

    Science.gov (United States)

    Paget, Sonia; Dubuissez, Marion; Dehennaut, Vanessa; Nassour, Joe; Harmon, Brennan T; Spruyt, Nathalie; Loison, Ingrid; Abbadie, Corinne; Rood, Brian R; Leprince, Dominique

    2017-01-10

    The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor mediating the p53-dependent apoptotic response to irreparable DNA double-strand breaks (DSBs) through direct transcriptional repression of SIRT1. HIC1 is also essential for DSB repair as silencing of endogenous HIC1 in BJ-hTERT fibroblasts significantly delays DNA repair in functional Comet assays. HIC1 SUMOylation favours its interaction with MTA1, a component of NuRD complexes. In contrast with irreparable DSBs induced by 16-hours of etoposide treatment, we show that repairable DSBs induced by 1 h etoposide treatment do not increase HIC1 SUMOylation or its interaction with MTA1. Furthermore, HIC1 SUMOylation is dispensable for DNA repair since the non-SUMOylatable E316A mutant is as efficient as wt HIC1 in Comet assays. Upon induction of irreparable DSBs, the ATM-mediated increase of HIC1 SUMOylation is independent of its effector kinase Chk2. Moreover, irreparable DSBs strongly increase both the interaction of HIC1 with MTA1 and MTA3 and their binding to the SIRT1 promoter. To characterize the molecular mechanisms sustained by this increased repression potential, we established global expression profiles of BJ-hTERT fibroblasts transfected with HIC1-siRNA or control siRNA and treated or not with etoposide. We identified 475 genes potentially repressed by HIC1 with cell death and cell cycle as the main cellular functions identified by pathway analysis. Among them, CXCL12, EPHA4, TGFβR3 and TRIB2, also known as MTA1 target-genes, were validated by qRT-PCR analyses. Thus, our data demonstrate that HIC1 SUMOylation is important for the transcriptional response to non-repairable DSBs but dispensable for DNA repair.

  15. Hypospadias repair

    Science.gov (United States)

    ... the problem. If the repair is not done, problems may occur later on such as: Difficulty controlling and directing urine stream A curve in the penis during erection Decreased fertility Embarrassment about appearance of penis Surgery ...

  16. Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair

    DEFF Research Database (Denmark)

    Smeenk, Godelieve; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate...... accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms...... for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance...

  17. Tel1 and Rif2 Regulate MRX Functions in End-Tethering and Repair of DNA Double-Strand Breaks.

    Directory of Open Access Journals (Sweden)

    Corinne Cassani

    2016-02-01

    Full Text Available The cellular response to DNA double-strand breaks (DSBs is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals, which recruits the checkpoint kinase Tel1/ATM to DSBs. In Saccharomyces cerevisiae, the role of Tel1 at DSBs remains enigmatic, as tel1Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a rad50-V1269M allele that sensitizes tel1Δ cells to genotoxic agents. The MRV1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, tel1Δ rad50-V1269M cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR or nonhomologous end joining (NHEJ. These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50.

  18. Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes.

    Directory of Open Access Journals (Sweden)

    Maria E Morales

    Full Text Available Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR and single strand annealing (SSA, which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the "error prone" non-homologous end joining (alt-NHEJ while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.

  19. Spp1, a member of the Set1 Complex, promotes meiotic DSB formation in promoters by tethering histone H3K4 methylation sites to chromosome axes.

    Science.gov (United States)

    Sommermeyer, Vérane; Béneut, Claire; Chaplais, Emmanuel; Serrentino, Maria Elisabetta; Borde, Valérie

    2013-01-10

    Meiotic chromosomes are organized into arrays of loops that are anchored to the chromosome axis structure. Programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination, catalyzed by Spo11 and accessory DSB proteins, form in loop sequences in promoters, whereas the DSB proteins are located on chromosome axes. Mechanisms bridging these two chromosomal regions for DSB formation have remained elusive. Here we show that Spp1, a conserved member of the histone H3K4 methyltransferase Set1 complex, is required for normal levels of DSB formation and is associated with chromosome axes during meiosis, where it physically interacts with the Mer2 DSB protein. The PHD finger module of Spp1, which reads H3K4 methylation close to promoters, promotes DSB formation by tethering these regions to chromosome axes and activating cleavage by the DSB proteins. This paper provides the molecular mechanism linking DSB sequences to chromosome axes and explains why H3K4 methylation is important for meiotic recombination.

  20. Femoral hernia repair

    Science.gov (United States)

    Femorocele repair; Herniorrhaphy; Hernioplasty - femoral ... During surgery to repair the hernia, the bulging tissue is pushed back in. The weakened area is sewn closed or strengthened. This repair ...

  1. Undescended testicle repair

    Science.gov (United States)

    Orchidopexy; Inguinal orchidopexy; Orchiopexy; Repair of undescended testicle; Cryptorchidism repair ... first year of life without treatment. Undescended testicle repair surgery is recommended for patients whose testicles do ...

  2. Erwinia carotovora DsbA mutants: evidence for a periplasmic-stress signal transduction system affecting transcription of genes encoding secreted proteins.

    Science.gov (United States)

    Vincent-Sealy, L V; Thomas, J D; Commander, P; Salmond, G P

    1999-08-01

    The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene

  3. Optical transmission radiation damage and recovery stimulation of DSB: Ce3+ inorganic scintillation material

    Science.gov (United States)

    Borisevich, A.; Dormenev, V.; Korjik, M.; Kozlov, D.; Mechinsky, V.; Novotny, R. W.

    2015-02-01

    Recently, a new scintillation material DSB: Ce3+ was announced. It can be produced in a form of glass or nano-structured glass ceramics with application of standard glass production technology with successive thermal annealing. When doped with Ce3+, material can be applied as scintillator. Light yield of scintillation is near 100 phe/MeV. Un-doped material has a wide optical window from 4.5eV and can be applied to detect Cherenkov light. Temperature dependence of the light yield LY(T) is 0.05% which is 40 times less than in case of PWO. It can be used for detectors tolerant to a temperature variation between -20° to +20°C. Several samples with dimensions of 15x15x7 mm3 have been tested for damage effects on the optical transmission under irradiation with γ-quanta. It was found that the induced absorption in the scintillation range depends on the doping concentration and varies in range of 0.5-7 m-1. Spontaneous recovery of induced absorption has fast initial component. Up to 25% of the damaged transmission is recuperated in 6 hours. Afterwards it remains practically constant if the samples are kept in the dark. However, induced absorption is reduced by a factor of 2 by annealing at 50°C and completely removed in a short time when annealing at 100°C. A significant acceleration of the induced absorption recovery is observed by illumination with visible and IR light. This effect is observed for the first time in a Ce-doped scintillation material. It indicates, that radiation induced absorption in DSB: Ce scintillation material can be retained at the acceptable level by stimulation with light in a strong irradiation environment of collider experiments.

  4. Robustness and Recovery in Train Scheduling - a simulation study from DSB S-tog a/s

    DEFF Research Database (Denmark)

    Hofman, Mads A.; Madsen, Line; Groth, Julie Jespersen

    This paper presents a simulation model to study the robustness of timetables of DSB S-tog a/s, the city rail of Copenhagen. Dealing with rush hour scenarios only, the simulation model investigates the effects of disturbances on the S-tog network. Several timetables are analyzed with respect to ro...

  5. Plan Repair using a Plan Library

    NARCIS (Netherlands)

    Van der Krogt, R.P.J.; De Weerdt, M.M.

    2005-01-01

    Plan library's have proven their added value to the efficiency of planning. In this paper, we present results on the use of a plan library to plan repair. We show that using a relatively simple library, we can already obtain significant improvements in efficiency compared to plan repair without a li

  6. Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    Science.gov (United States)

    Singh, Satyendra K.; Wang, Minli; Staudt, Christian; Iliakis, George

    2011-01-01

    In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar–phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of ‘naked’ or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8–24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization. PMID:21745815

  7. A role for the malignant brain tumour (MBT domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

    Directory of Open Access Journals (Sweden)

    Nicholas M Johnson

    Full Text Available Malignant brain tumour (MBT domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR for the repair of DNA double-strand breaks (DSBs. lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

  8. DNA repair and cytokines: TGF-beta, IL-6, and thrombopoietin as different biomarkers of radioresistance

    Directory of Open Access Journals (Sweden)

    Francesca Bianca Aiello

    2016-07-01

    Full Text Available Double strand breaks (DSBs induced by radiotherapy are highly cytotoxic lesions, leading to chromosomal aberrations and cell death. ATM-dependent DNA-damage response, non-homologous end joining, and homologous recombination pathways coordinately contribute to repairing DSBs in higher eukaryotes. It is known that the expression of DSB repair genes is increased in tumors which is one of the main reasons for radioresistance. The inhibition of DSB repair pathways may be useful to increase tumor cell radiosensitivity and may target stem cell-like cancer cells, known to be the most radioresistant tumor components. Commonly overexpressed in neoplastic cells, cytokines confer radioresistance by promoting proliferation, survival, invasion, and angiogenesis. Unfortunately, tumor irradiation increases the expression of various cytokines displaying these effects, including transforming growth factor-beta and interlukin-6. Recently the capabilities of these cytokines to support DNA repair pathways and the ATM-dependent DNA response have been demonstrated. Thrombopoietin, essential for megakaryopoiesis and very important for hematopoietic stem cell homeostasis, has also been found to promote DNA repair in a highly selective manner. These findings reveal a novel mechanism underlying cytokine-related radioresistance, which may be clinically relevant. Therapies targeting specific cytokines may be used to improve radiosensitivity. Specific inhibitors may be chosen in consideration of different tumor microenvironments. Thrombopoietin may be useful in fending off irradiation-induced loss of hematopoietic stem cells.

  9. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

    Energy Technology Data Exchange (ETDEWEB)

    Dupuy, Aurélie [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Sarasin, Alain, E-mail: alain.sarasin@gustaveroussy.fr [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Service de Génétique, Institut Gustave Roussy (France)

    2015-06-15

    Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  10. Double-strand break repair processes drive evolution of the mitochondrial genome in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Shedge Vikas

    2011-09-01

    Full Text Available Abstract Background The mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining (NHEJ activities producing variability in size and organization. Plant mitochondrial DNA also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. Arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene MSH1 (MutS 1 homolog, lending itself to detailed study of de novo mitochondrial genome activity. In the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution. Results We obtained evidence of double-strand break (DSB repair, including NHEJ, sequence deletions and mitochondrial asymmetric recombination activity in Arabidopsis wild-type and msh1 mutants on the basis of data generated by Illumina deep sequencing and confirmed by DNA gel blot analysis. On a larger scale, with mitochondrial comparisons across 72 Arabidopsis ecotypes, similar evidence of DSB repair activity differentiated ecotypes. Forty-seven repeat pairs were active in DNA exchange in the msh1 mutant. Recombination sites showed asymmetrical DNA exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. De novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. Substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes. Conclusions Extensive mitochondrial genomic variation within a single plant species derives largely from DSB activity and its repair. Observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. On a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants.

  11. Intestinal obstruction repair

    Science.gov (United States)

    Repair of volvulus; Intestinal volvulus - repair; Bowel obstruction - repair ... Intestinal obstruction repair is done while you are under general anesthesia . This means you are asleep and DO NOT feel pain. ...

  12. Aortic aneurysm repair - endovascular

    Science.gov (United States)

    EVAR; Endovascular aneurysm repair - aorta; AAA repair - endovascular; Repair - aortic aneurysm - endovascular ... Endovascular aortic repair is done because your aneurysm is very large, growing quickly, or is leaking or bleeding. You may have ...

  13. Motorcycle Repair.

    Science.gov (United States)

    Hein, Jim; Bundy, Mike

    This motorcycle repair curriculum guide contains the following ten areas of study: brake systems, clutches, constant mesh transmissions, final drives, suspension, mechanical starting mechanisms, electrical systems, fuel systems, lubrication systems, and overhead camshafts. Each area consists of one or more units of instruction. Each instructional…

  14. Topoisomerase II-mediated DNA damage is differently repaired during the cell cycle by non-homologous end joining and homologous recombination.

    Directory of Open Access Journals (Sweden)

    Marcelo de Campos-Nebel

    Full Text Available Topoisomerase II (Top2 is a nuclear enzyme involved in several metabolic processes of DNA. Chemotherapy agents that poison Top2 are known to induce persistent protein-mediated DNA double strand breaks (DSB. In this report, by using knock down experiments, we demonstrated that Top2alpha was largely responsible for the induction of gammaH2AX and cytotoxicity by the Top2 poisons idarubicin and etoposide in normal human cells. As DSB resulting from Top2 poisons-mediated damage may be repaired by non-homologous end joining (NHEJ or homologous recombination (HR, we aimed to analyze both DNA repair pathways. We found that DNA-PKcs was rapidly activated in human cells, as evidenced by autophosphorylation at serine 2056, following Top2-mediated DNA damage. The chemical inhibition of DNA-PKcs by wortmannin and vanillin resulted in an increased accumulation of DNA DSB, as evaluated by the comet assay. This was supported by a hypersensitive phenotype to Top2 poisons of Ku80- and DNA-PKcs- defective Chinese hamster cell lines. We also showed that Rad51 protein levels, Rad51 foci formation and sister chromatid exchanges were increased in human cells following Top2-mediated DNA damage. In support, BRCA2- and Rad51C- defective Chinese hamster cells displayed hypersensitivity to Top2 poisons. The analysis by immunofluorescence of the DNA DSB repair response in synchronized human cell cultures revealed activation of DNA-PKcs throughout the cell cycle and Rad51 foci formation in S and late S/G2 cells. Additionally, we found an increase of DNA-PKcs-mediated residual repair events, but not Rad51 residual foci, into micronucleated and apoptotic cells. Therefore, we conclude that in human cells both NHEJ and HR are required, with cell cycle stage specificity, for the repair of Top2-mediated reversible DNA damage. Moreover, NHEJ-mediated residual repair events are more frequently associated to irreversibly damaged cells.

  15. CrowdAidRepair: A Crowd-Aided Interactive Data Repairing Method

    KAUST Repository

    Zhou, Jian

    2016-03-25

    Data repairing aims at discovering and correcting erroneous data in databases. Traditional methods relying on predefined quality rules to detect the conflict between data may fail to choose the right way to fix the detected conflict. Recent efforts turn to use the power of crowd in data repairing, but the crowd power has its own drawbacks such as high human intervention cost and inevitable low efficiency. In this paper, we propose a crowd-aided interactive data repairing method which takes the advantages of both rule-based method and crowd-based method. Particularly, we investigate the interaction between crowd-based repairing and rule-based repairing, and show that by doing crowd-based repairing to a small portion of values, we can greatly improve the repairing quality of the rule-based repairing method. Although we prove that the optimal interaction scheme using the least number of values for crowd-based repairing to maximize the imputation recall is not feasible to be achieved, still, our proposed solution identifies an efficient scheme through investigating the inconsistencies and the dependencies between values in the repairing process. Our empirical study on three data collections demonstrates the high repairing quality of CrowdAidRepair, as well as the efficiency of the generated interaction scheme over baselines.

  16. Turbine repair process, repaired coating, and repaired turbine component

    Energy Technology Data Exchange (ETDEWEB)

    Das, Rupak; Delvaux, John McConnell; Garcia-Crespo, Andres Jose

    2015-11-03

    A turbine repair process, a repaired coating, and a repaired turbine component are disclosed. The turbine repair process includes providing a turbine component having a higher-pressure region and a lower-pressure region, introducing particles into the higher-pressure region, and at least partially repairing an opening between the higher-pressure region and the lower-pressure region with at least one of the particles to form a repaired turbine component. The repaired coating includes a silicon material, a ceramic matrix composite material, and a repaired region having the silicon material deposited on and surrounded by the ceramic matrix composite material. The repaired turbine component a ceramic matrix composite layer and a repaired region having silicon material deposited on and surrounded by the ceramic matrix composite material.

  17. Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context.

    Science.gov (United States)

    Gunn, Amanda; Bennardo, Nicole; Cheng, Anita; Stark, Jeremy M

    2011-12-09

    During repair of multiple chromosomal double strand breaks (DSBs), matching the correct DSB ends is essential to limit rearrangements. To investigate the maintenance of correct end use, we examined repair of two tandem noncohesive DSBs generated by endonuclease I-SceI and the 3' nonprocessive exonuclease Trex2, which can be expressed as an I-SceI-Trex2 fusion. We examined end joining (EJ) repair that maintains correct ends (proximal-EJ) versus using incorrect ends (distal-EJ), which provides a relative measure of incorrect end use (distal end use). Previous studies showed that ATM is important to limit distal end use. Here we show that DNA-PKcs kinase activity and RAD50 are also important to limit distal end use, but that H2AX is dispensable. In contrast, we find that ATM, DNA-PKcs, and RAD50 have distinct effects on repair events requiring end processing. Furthermore, we developed reporters to examine the effects of the transcription context on DSB repair, using an inducible promoter. We find that a DSB downstream from an active promoter shows a higher frequency of distal end use, and a greater reliance on ATM for limiting incorrect end use. Conversely, DSB transcription context does not affect end processing during EJ, the frequency of homology-directed repair, or the role of RAD50 and DNA-PKcs in limiting distal end use. We suggest that RAD50, DNA-PKcs kinase activity, and transcription context are each important to limit incorrect end use during EJ repair of multiple DSBs, but that these factors and conditions have distinct roles during repair events requiring end processing.

  18. Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre

    DEFF Research Database (Denmark)

    Lisby, M.; Mortensen, Uffe Hasbro; Rothstein, R.

    2003-01-01

    DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize...... in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint......-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB....

  19. Development of a radiation track structure clustering algorithm for the prediction of DNA DSB yields and radiation induced cell death in Eukaryotic cells

    Science.gov (United States)

    Douglass, Michael; Bezak, Eva; Penfold, Scott

    2015-04-01

    The preliminary framework of a combined radiobiological model is developed and calibrated in the current work. The model simulates the production of individual cells forming a tumour, the spatial distribution of individual ionization events (using Geant4-DNA) and the stochastic biochemical repair of DNA double strand breaks (DSBs) leading to the prediction of survival or death of individual cells. In the current work, we expand upon a previously developed tumour generation and irradiation model to include a stochastic ionization damage clustering and DNA lesion repair model. The Geant4 code enabled the positions of each ionization event in the cells to be simulated and recorded for analysis. An algorithm was developed to cluster the ionization events in each cell into simple and complex double strand breaks. The two lesion kinetic (TLK) model was then adapted to predict DSB repair kinetics and the resultant cell survival curve. The parameters in the cell survival model were then calibrated using experimental cell survival data of V79 cells after low energy proton irradiation. A monolayer of V79 cells was simulated using the tumour generation code developed previously. The cells were then irradiated by protons with mean energies of 0.76 MeV and 1.9 MeV using a customized version of Geant4. By replicating the experimental parameters of a low energy proton irradiation experiment and calibrating the model with two sets of data, the model is now capable of predicting V79 cell survival after low energy (<2 MeV) proton irradiation for a custom set of input parameters. The novelty of this model is the realistic cellular geometry which can be irradiated using Geant4-DNA and the method in which the double strand breaks are predicted from clustering the spatial distribution of ionisation events. Unlike the original TLK model which calculates a tumour average cell survival probability, the cell survival probability is calculated for each cell in the geometric tumour model

  20. Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

    Directory of Open Access Journals (Sweden)

    Antoine Marmignon

    2014-08-01

    Full Text Available During somatic differentiation, physiological DNA double-strand breaks (DSB can drive programmed genome rearrangements (PGR, during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES. IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium

  1. Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

    Science.gov (United States)

    Marmignon, Antoine; Bischerour, Julien; Silve, Aude; Fojcik, Clémentine; Dubois, Emeline; Arnaiz, Olivier; Kapusta, Aurélie; Malinsky, Sophie; Bétermier, Mireille

    2014-08-01

    During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage

  2. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    Science.gov (United States)

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  3. Base excision repair efficiency and mechanism in nuclear extracts are influenced by the ratio between volume of nuclear extraction buffer and nuclei-Implications for comparative studies

    DEFF Research Database (Denmark)

    Akbari, Mansour; Krokan, Hans E

    2012-01-01

    The base excision repair (BER) pathway corrects many different DNA base lesions and is important for genomic stability. The mechanism of BER cannot easily be investigated in intact cells and therefore in vitro methods that reflect the in vivo processes are in high demand. Reconstitution of BER...... using purified proteins essentially mirror properties of the proteins used, and does not necessarily reflect the mechanism as it occurs in the cell. Nuclear extracts from cultured cells have the capacity to carry out complete BER and can give important information on the mechanism. Furthermore...

  4. 无张力疝修补术治疗腹股沟疝的效果观察%Observation the Treatment Efficiency of Tension-free Hernia Repair in Inguinal Hernia

    Institute of Scientific and Technical Information of China (English)

    马庆国

    2015-01-01

    目的:观察无张力疝修补术对腹股沟疝的临床治疗效果。方法搜集2012年2月~2014年2月我院接收的腹股沟疝78例患者,按照不同手术方法分为研究组与对照组。对照组39例施行传统修补术,研究组39例施行无张力疝修补术。观察研究组与对照组的临床效果,并对比。结果与对照组相比,研究组术中出血量较少,手术时间较短,并发症和复发较少,有明显差异,有统计学意义(P<0.05)。结论无张力疝修补术对腹股沟疝的治疗效果较好,出血少,恢复快,且并发症和复发少。%Objective Treatment effect of tension-free hernia repair in inguinal hernia is to be observed. Methods Choose 78 patients with inguinal hernia who are treated in hospital from February 2012 to February 2014 and separate them into study group and control group according to different surgical treatment methods.39 patients in control group are given conventional repair treatment,while,39 patients in study group are given tension-free hernia repair treatment,and then observe and compare the clinical treatment efficiency of the two groups. Results Compared to control group,the hemorrhage amount and the surgical treatment time in study group is much less,and what’s more,the patients in study group has less complication incidence and reoccurrence rate.There is an efficiency difference between two groups and such a differential has statistic value(P<0.05). Conclusion Tension-free hernia repair treatment is quite effective to cure inguinal hernia with less hemorrhage and complications,and in addition,it is conducive to recovery of patients,therefore,it is worthy of being applied and promoted.

  5. INCREASING EFFICIENCY OF REPAIRING, MANUFACTURING AND OPERATION OF THE TPP FACILITIES BY TECHNOLOGY OF GAS-THERMAL COATING AND LASER SURFACE MELTING

    Directory of Open Access Journals (Sweden)

    O. E. Grachev

    2015-01-01

    Full Text Available The article considers effectiveness increase of the TPP heat-mechanical equipment repair, manufacturing and maintenance as exemplified by gas-thermal technique for hardening laststages rotor blades of the steam turbines. The rotor blades work under conditions of intense power loading, their airfoil being erosion-corrosion destructed by the action of the moist-steam flow. Repairing companies employ quite a number of technologies to restore some of erosion-worn rotor blades. Inter alia, argon-arc, plasma and gas-powder weld deposition of the original material with subsequent machining, stellite protection recovery, electrical spark alloying the entry edge mat surface, spraying ion-plasma coating on the blade airfoil surface. In domestic turbine building, rotor blades of the steam turbines last stages are manufactured of martensitic class stainless steel. The key condition for successful blade restoration is thermal effect minimizing on the base material for excluding the slag areas possible forming. The laser surface coating technology provides these conditions. They coat the surface of an item being processed by way of melting the base and the adding material. In as much the base melts smallest, the coating characteristics depend mainly on the properties of adding material. The procedure of laser coating passes through several stages including physical contact creation, chemical interaction (laser radiation absorption, volumetrical processes resulting in formation of stable bonds in volume of the materials that have reacted. For the low-pressure cylinder rotor blades supplementary protection against erosion destruction, LLC ‘Technological Systems of Protective Coating’ developed technology of the blade airfoil protective finish by method of high-speed gas-flame sputter. The company realized this technology in 2012 during K-200-12,8 turbine (of the Leningrad Metallurgical Works – LMZ repairing in Zainsk SDPP by JSC ‘Tatenergo’. The

  6. REPAIR TECHNOLOGY IMPROVEMENT OF SPECIALIZED FREIGHT CARS

    Directory of Open Access Journals (Sweden)

    V. M. Bubnov

    2016-02-01

    Full Text Available Purpose. The volume of cargo transportation demands the introduction of a new generation of cars that would be able to provide all the needs of carriers. But this is impossible without the implementation of renovation repair facilities with the introduction of new technologies and modernization of the repair process. Repair of rolling stock is a key factor that must proceed with the establishment of new cars, as not all of the inventions may be repaired in car-repair depots, most of which are obsolete. The purpose is to analyze the possibility of increasing the efficiency of the repair process by introducing new repair technologies or improving the existing ones. It will improve not only the quality of the repair, but also its rate. Methodology. Works on improving the designs of freight cars are held by many design organizations in almost all industrialized countries. It makes repair organizations (depots and car-repair plants to upgrade the repair process. Achievements of-this goal is possible by improving the technology renovation and reorganization through the use of flexible flow technologies, which to date are the most effective in the repair of rolling stock. Findings. When performing the analysis it was determined that there are different designs of cars. More of cars are all-purpose and their repair does not cause difficulties for car-repair business. However, the number of specialized cars is also significant, and the technology of their repair should be improved. One of the reasons is that many models, such as tank wagons for the carriage of sulfur, are intended for the carriage of dangerous goods and their failure at the time of motion is not permitted. Originality. Firstly the authors have defined direction at improving technologies of repair specialized cars. Practical value. Actual improvement in the construction of cars is to improve the existing repair facilities. In addition, the repair technology using nowadays when repairing

  7. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    Energy Technology Data Exchange (ETDEWEB)

    Bajinskis, Ainars, E-mail: ainars.bajinskis@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden); Olsson, Gunilla; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden)

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO{sub 3}). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  8. Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism

    OpenAIRE

    Grabowska, AD; Wandel, MP; Łasica, AM; Nesteruk, M; Roszczenko, P; Wyszyńska, A; Godlewska, R; Jagusztyn-Krynicka, EK

    2011-01-01

    Abstract Background Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in Campyl...

  9. Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Campos-Nebel, Marcelo de [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)], E-mail: mnebel@hematologia.anm.edu.ar; Larripa, Irene; Gonzalez-Cid, Marcela [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)

    2008-11-10

    Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by {gamma}H2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU

  10. DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids

    Directory of Open Access Journals (Sweden)

    Emad A. Ahmed

    2015-12-01

    Full Text Available Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX foci marking DNA double strand breaks (DSBs in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP-ribose polymerase 1 (PARP1 inhibitor (DPQ-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

  11. DNA double strand break repair enzymes function at multiple steps in retroviral infection

    Directory of Open Access Journals (Sweden)

    Agematsu Kazunaga

    2009-12-01

    Full Text Available Abstract Background DNA double strand break (DSB repair enzymes are thought to be necessary for retroviral infection, especially for the post-integration repair and circularization of viral cDNA. However, the detailed roles of DSB repair enzymes in retroviral infection remain to be elucidated. Results A GFP reporter assay showed that the infectivity of an HIV-based vector decreased in ATM- and DNA-PKcs-deficient cells when compared with their complemented cells, while that of an MLV-based vector was diminished in Mre11- and DNA-PKcs-deficient cells. By using a method based on inverse- and Alu-PCR, we analyzed sequences around 3' HIV-1 integration sites in ATM-, Mre11- and NBS1- deficient cells. Increased abnormal junctions between the HIV-1 provirus and the host DNA were found in these mutant cell lines compared to the complemented cell lines and control MRC5SV cells. The abnormal junctions contained two types of insertions: 1 GT dinucleotides, which are normally removed by integrase during integration, and 2 inserted nucleotides of unknown origin. Artemis-deficient cells also showed such abnormalities. In Mre11-deficient cells, part of a primer binding site sequence was also detected. The 5' host-virus junctions in the mutant cells also contained these types of abnormal nucleotides. Moreover, the host-virus junctions of the MLV provirus showed similar abnormalities. These findings suggest that DSB repair enzymes play roles in the 3'-processing reaction and protection of the ends of viral DNA after reverse transcription. We also identified both 5' and 3' junctional sequences of the same provirus by inverse PCR and found that only the 3' junctions were abnormal with aberrant short repeats, indicating that the integration step was partially impaired in these cells. Furthermore, the conserved base preferences around HIV-1 integration sites were partially altered in ATM-deficient cells. Conclusions These results suggest that DSB repair enzymes are

  12. Eye muscle repair - discharge

    Science.gov (United States)

    ... Lazy eye repair - discharge; Strabismus repair - discharge; Extraocular muscle surgery - discharge ... You or your child had eye muscle repair surgery to correct eye muscle ... term for crossed eyes is strabismus. Children most often ...

  13. Ventral hernia repair

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/007661.htm Ventral hernia repair To use the sharing features on this page, please enable JavaScript. Ventral hernia repair is surgery to repair a ventral hernia. ...

  14. Brain aneurysm repair

    Science.gov (United States)

    ... aneurysm repair; Dissecting aneurysm repair; Endovascular aneurysm repair - brain; Subarachnoid hemorrhage - aneurysm ... Your scalp, skull, and the coverings of the brain are opened. A metal clip is placed at ...

  15. Three Kinds of Crown Repairing Marginal Fit Efficiency Comparing%三种全冠修复边缘适合性效果对比

    Institute of Scientific and Technical Information of China (English)

    张晓正

    2014-01-01

    Objective To repairing ef ects on marginal adaptation of Ni-Cr al oy porcelain crowns,al-ceramic crowns and metal ceramic crowns compared to guide clinical treatment. Methods According to the teeth conditions and needs of patients,use Ni-Cr al oy porcelain teeth,dental precious metal ceramic and al -ceramic dental restoration to repair,one year after referral to observe al the objects in gingival color,gingival index and marginal fit. Results Ni-Cr al oy porcelain teeth marginal fit of Class A accounted for 86.5%.accounting for 95.1%of Class A of the marginal fit of precious metal porcelain tooth edge.al -cerainlc dental marginal fit of Class A accounted for 96.6%. Conclusion For the Ni-Cr al oy porcelain teeth,crowns fit is poor and the binder dissolution caused by the local pigment deposi-tion is the main reason to cause periodontal damage caused by gum color change.Al-ceramic dental and precious metal ceramic teeth has go d biocompatibility and marginal adaptation of virtual y no efect on the color of the gums and gingival index.%目的:对比三种全冠修复的边缘适合性效果进行对比,以指导临床的治疗。方法根据临床完成的镍铬合金烤瓷冠、贵金属烤瓷冠和全瓷冠修复,1年后复诊观察所有全冠的边缘适合性。结果镍铬合金烤瓷牙边缘适合性A级的占86.5%,贵金属烤瓷牙边缘适合性A级的占95.1%,全瓷牙边缘适合性 A级的占96.6%。结论对于镍铬合金烤瓷冠边缘的密合性差,而全瓷冠与贵金属烤瓷冠具有很好的边缘适合性。

  16. Scarf Repair of Composite Laminates

    Directory of Open Access Journals (Sweden)

    Xie Zonghong

    2016-01-01

    Full Text Available The use of composite materials, such as carbon-fiber reinforced plastic (CFRP composites, aero-structures has led to an increased need of advanced assembly joining and repair technologies. Adhesive bonded repairs as an alternative to recover full or part of initial strength were investigated. Tests were conducted with the objective of evaluating the effectiveness of techniques used for repairing damage fiber reinforced laminated composites. Failure loads and failure modes were generated and compared with the following parameters: scarf angles, roughness of grind tool and number of external plies. Results showed that scarf angle was the critical parameter and the largest tensile strength was observed with the smallest scarf angle. Besides, the use of external plies at the outer surface could not increase the repairs efficiency for large scarf angle. Preparing the repair surfaces by sanding them with a sander ranging from 60 to 100 grit number had significant effect on the failure load. These results allowed the proposal of design principles for repairing CFRP structures.

  17. IGF-1R inhibition enhances radiosensitivity and delays double-strand break repair by both non-homologous end-joining and homologous recombination.

    Science.gov (United States)

    Chitnis, M M; Lodhia, K A; Aleksic, T; Gao, S; Protheroe, A S; Macaulay, V M

    2014-11-06

    Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) enhances tumor cell sensitivity to ionizing radiation. It is not clear how this effect is mediated, nor whether this approach can be applied effectively in the clinic. We previously showed that IGF-1R depletion delays repair of radiation-induced DNA double-strand breaks (DSBs), unlikely to be explained entirely by reduction in homologous recombination (HR) repair. The current study tested the hypothesis that IGF-1R inhibition induces a repair defect that involves non-homologous end joining (NHEJ). IGF-1R inhibitor AZ12253801 blocked cell survival and radiosensitized IGF-1R-overexpressing murine fibroblasts but not isogenic IGF-1R-null cells, supporting specificity for IGF-1R. IGF-1R inhibition enhanced radiosensitivity in DU145, PC3 and 22Rv1 prostate cancer cells, comparable to effects of Ataxia Telangiectasia Mutated inhibition. AZ12253801-treated DU145 cells showed delayed resolution of γH2AX foci, apparent within 1 h of irradiation and persisting for 24 h. In contrast, IGF-1R inhibition did not influence radiosensitivity or γH2AX focus resolution in LNCaP-LN3 cells, suggesting that radiosensitization tracks with the ability of IGF-1R to influence DSB repair. To differentiate effects on repair from growth and cell-survival responses, we tested AZ12253801 in DU145 cells at sub-SF50 concentrations that had no early (⩽48 h) effects on cell cycle distribution or apoptosis induction. Irradiated cultures contained abnormal mitoses, and after 5 days IGF-1R-inhibited cells showed enhanced radiation-induced polyploidy and nuclear fragmentation, consistent with the consequences of entry into mitosis with incompletely repaired DNA. AZ12253801 radiosensitized DNA-dependent protein kinase (DNA-PK)-proficient but not DNA-PK-deficient glioblastoma cells, and did not radiosensitize DNA-PK-inhibited DU145 cells, suggesting that in the context of DSB repair, IGF-1R functions in the same pathway as DNA

  18. Homologous recombination in DNA repair and DNA damage tolerance

    Institute of Scientific and Technical Information of China (English)

    Xuan Li; Wolf-Dietrich Heyer

    2008-01-01

    Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical sup-port for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modaUties of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

  19. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

    Science.gov (United States)

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M; Samuel, Glady Hazitha; Anderson, Michelle A E; Dahlem, Timothy J; Myles, Kevin M; Adelman, Zach N

    2015-03-31

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

  20. The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination.

    Directory of Open Access Journals (Sweden)

    Rebecca C Burgess

    Full Text Available Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR. Although its activity has been implicated in several steps of HR, its exact role(s at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA. This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box of Rad54 (Rad54-AA. Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

  1. Recombinational DNA repair and human disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Larry H.; Schild, David

    2002-11-30

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

  2. c-Myc Suppression of DNA Double-strand Break Repair12

    Science.gov (United States)

    Li, Zhaozhong; Owonikoko, Taofeek K; Sun, Shi-Yong; Ramalingam, Suresh S; Doetsch, Paul W; Xiao, Zhi-Qiang; Khuri, Fadlo R; Curran, Walter J; Deng, Xingming

    2012-01-01

    c-Myc is a transcriptional factor that functions as a central regulator of cell growth, proliferation, and apoptosis. Overexpression of c-Myc also enhances DNA double-strand breaks (DSBs), genetic instability, and tumorigenesis. However, the mechanism(s) involved remains elusive. Here, we discovered that γ-ray ionizing radiation-induced DSBs promote c-Myc to form foci and to co-localize with γ-H2AX. Conditional expression of c-Myc in HO15.19 c-Myc null cells using the Tet-Off/Tet-On inducible system results in down-regulation of Ku DNA binding and suppressed activities of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and DNA end-joining, leading to inhibition of DSB repair and enhanced chromosomal and chromatid breaks. Expression of c-Myc reduces both signal and coding joins with decreased fidelity during V(D)J recombination. Mechanistically, c-Myc directly interacts with Ku70 protein through its Myc box II (MBII) domain. Removal of the MBII domain from c-Myc abrogates its inhibitory effects on Ku DNA binding, DNA-PKcs, and DNA end-joining activities, which results in loss of c-Myc's ability to block DSB repair and V(D)J recombination. Interestingly, c-Myc directly disrupts the Ku/DNA-PKcs complex in vitro and in vivo. Thus, c-Myc suppression of DSB repair and V(D)J recombination may occur through inhibition of the nonhomologous end-joining pathway, which provides insight into the mechanism of c-Myc in the development of tumors through promotion of genomic instability. PMID:23308051

  3. H. pylori-Induced DNA Strand Breaks Are Introduced by Nucleotide Excision Repair Endonucleases and Promote NF-κB Target Gene Expression

    Directory of Open Access Journals (Sweden)

    Mara L. Hartung

    2015-10-01

    Full Text Available The human bacterial pathogen Helicobacter pylori exhibits genotoxic properties that promote gastric carcinogenesis. H. pylori introduces DNA double strand breaks (DSBs in epithelial cells that trigger host cell DNA repair efforts. Here, we show that H. pylori-induced DSBs are repaired via error-prone, potentially mutagenic non-homologous end-joining. A genome-wide screen for factors contributing to DSB induction revealed a critical role for the H. pylori type IV secretion system (T4SS. Inhibition of transcription, as well as NF-κB/RelA-specific RNAi, abrogates DSB formation. DSB induction further requires β1-integrin signaling. DSBs are introduced by the nucleotide excision repair endonucleases XPF and XPG, which, together with RelA, are recruited to chromatin in a highly coordinated, T4SS-dependent manner. Interestingly, XPF/XPG-mediated DNA DSBs promote NF-κB target gene transactivation and host cell survival. In summary, H. pylori induces XPF/XPG-mediated DNA damage through activation of the T4SS/β1-integrin signaling axis, which promotes NF-κB target gene expression and host cell survival.

  4. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

    Science.gov (United States)

    Peterson, S R; Kurimasa, A; Oshimura, M; Dynan, W S; Bradbury, E M; Chen, D J

    1995-04-11

    The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

  5. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, S.R. [Los Alamos National Lab., NM (United States)]|[Tottori Univ., Yonago (Japan); Kurimasa, Akihiro; Oshimura, Mitsuo [Tottori Univ., Yonago (Japan); Dynan, W.S. [Univ. of Colorado, Boulder, CO (United States); Bradbury, E.M. [Los Alamos National Lab., NM (United States)]|[Univ. of California, Davis, CA (United States); Chen, D.J. [Los Alamos National Lab., NM (United States)

    1995-04-11

    The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an {approx}350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway. 38 refs., 3 figs.

  6. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae.

    Science.gov (United States)

    Stein, Alexis; Kalifa, Lidza; Sia, Elaine A

    2015-11-01

    Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.

  7. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

    OpenAIRE

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M.; Samuel, Glady Hazitha; Anderson, Michelle A.E.; Dahlem, Timothy J.; Myles, Kevin M.; Adelman, Zach N.

    2015-01-01

    Mosquitoes are vectors of both parasites and viruses responsible for high-impact diseases including malaria, dengue, and chikungunya. Novel interventions based on genetic modification of the mosquito genome are currently being developed and implemented. To comprehensively exploit such interventions, detailed knowledge of mosquito physiology, genetics, and genome engineering are required. We developed and validated a two-step process for performing high-efficiency site-specific insertion of ge...

  8. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

    DEFF Research Database (Denmark)

    Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs......) orchestrates and regulates cellular responses to DSBs at multiple levels, often involving extensive crosstalk between these modifications. Recent findings have revealed compelling insights into the complex mechanisms by which ubiquitin and UBLs regulate protein interactions with DSB sites to promote accurate...

  9. DSB-600A卫星电视接收机电路分析与故障检修

    Institute of Scientific and Technical Information of China (English)

    李育林; 雷新盛; 陈吉利

    2003-01-01

    @@ 1 DSB-600A卫星电视接收机电路分析 空中的卫星电视信号通过卫星接收天线接收和高频头下变频,变换为950~1 450 MHz的第一中频信号,由同轴电缆输送到卫星电视接收机的第二变频器,变频为510 MHz第二中频信号.

  10. Sent packing: protein engineering generates a new crystal form of Pseudomonas aeruginosa DsbA1 with increased catalytic surface accessibility

    Energy Technology Data Exchange (ETDEWEB)

    McMahon, Roisin M., E-mail: r.mcmahon1@uq.edu.au; Coinçon, Mathieu; Tay, Stephanie; Heras, Begoña [University of Queensland, 306 Carmody Road, Brisbane, Queensland 4072 (Australia); Morton, Craig J. [Biota Holdings Limited, Unit 10, 585 Blackburn Road, Notting Hill, Victoria 3168 (Australia); Scanlon, Martin J. [Monash University, 381 Royal Parade, Parkville, Victoria 3052 (Australia); Martin, Jennifer L. [University of Queensland, 306 Carmody Road, Brisbane, Queensland 4072 (Australia)

    2015-11-26

    The crystal structure of a P. aeruginosa DsbA1 variant is more suitable for fragment-based lead discovery efforts to identify inhibitors of this antimicrobial drug target. In the reported structures the active site of the protein can simultaneously bind multiple ligands introduced in the crystallization solution or via soaking. Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.

  11. DNA double-strand break repair: a theoretical framework and its application.

    Science.gov (United States)

    Murray, Philip J; Cornelissen, Bart; Vallis, Katherine A; Chapman, S Jon

    2016-01-01

    DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γH2AX. Many copies of γH2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti-γH2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo. Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, (111)In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti-γH2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti-γH2AX-TAT and γH2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti-γH2AX antibody is labelled with Auger electron-emitting (111)In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti-γH2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti-γH2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage

  12. Induction of DNA DSB and its rejoining in clamped and non-clamped tumours after exposure to carbon ion beams in comparison to X rays.

    Science.gov (United States)

    Hirayama, R; Uzawa, A; Matsumoto, Y; Noguchi, M; Kase, Y; Takase, N; Ito, A; Koike, S; Ando, K; Okayasu, R; Furusawa, Y

    2011-02-01

    We studied double-strand breaks (DSB) induction and rejoining in clamped and non-clamped transplanted tumours in mice leg after exposure to 80 keV µm(-1) carbon ions and X rays. The yields of DSB in the tumours were analysed by a static-field gel electrophoresis. The OER of DSB after X rays was 1.68±0.31, and this value was not changed after 1 h rejoining time (1.40±0.26). These damages in oxygenated conditions were rejoined 60-70% within 1 h in situ. No difference was found between the exposure to X rays and carbon ions for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after exposure to carbon ions were similar to those after X rays, and the calculated relative biological effectivenesses of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not on the types of radiation quality.

  13. A Nuclei Isolation-Filter Elution Assay for the Simultaneous Detection of DNA SSB and DSB of Compact Plant Donor%核分离-滤膜洗脱技术同时检测植物DNA SSB与DSB

    Institute of Scientific and Technical Information of China (English)

    刘晓; 赵玉芳; 凌备备

    2000-01-01

    采用核分离-滤膜洗脱技术,将分离的细胞核在微孔滤膜上裂解后相继用中性溶液和碱性溶液洗脱,研究了60Co-γ辐射引发的大麦种胚细胞DNA断裂.结果显示,50Gy的60Co-γ辐射主要引发DNA单链断裂(DNA SSB);50~100 Gy的辐射引发的DNA断裂包括单链断裂(SSB)与双链断裂(DSB);当辐射剂量大于100 Gy时,DNA断裂的增加部分则以DSB为主.所用的实验方法适用于质地致密的植物材料并可在一次实验中同时检测DNA的SSB与DSB,为植物细胞的DNA链断裂作为生物标志物应用于环境诱变因子监测提供了基础.

  14. Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma

    Directory of Open Access Journals (Sweden)

    Koji eYoshimoto

    2012-12-01

    Full Text Available Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma. Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT has been described as the main modulator to determine the sensitivity of glioblastoma to TMZ, a subset of glioblastoma does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR, and the base-excision repair (BER pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break (SSB repair and double-strand break (DSB repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potent