DNA Double-strand Break Repair Mechanisms Research Articles

Smart Choice between Two DNA Double-strand Break Repair Mechanisms

Smart Choice between Two DNA Double-strand Break Repair Mechanisms

Differences in 53BP1 and BRCA1 regulation between cycling and non-cycling cells

BRCA1 and 53BP1 play decisive roles in the choice of DNA double-strand break repair mechanisms. BRCA1 promotes DNA end resection and homologous recombination (HR) during S/G2 phases of the cell cycle, while 53BP1 inhibits end resection and facilitates non-homologous end-joining (NHEJ), primarily during G1. This competitive relationship is critical for genome integrity during cell division. However, their relationship in the many cells in our body that are not cycling is unknown. We discovered profound differences in 53BP1 and BRCA1 regulation between cycling and non-cycling cells. Cellular growth arrest results in transcriptional downregulation of BRCA1 and activation of cathepsin-L (CTSL)-mediated degradation of 53BP1. Accordingly, growth-arrested cells do not form BRCA1 or 53BP1 ionizing radiation-induced foci (IRIF). Interestingly, cell cycle re-entry reverts this scenario, with upregulation of BRCA1, downregulation of CTSL, stabilization of 53BP1, and 53BP1 IRIF formation throughout the cycle, indicating that BRCA1 and 53BP1 are important in replicating cells and dispensable in non-cycling cells. We show that CTSL-mediated degradation of 53BP1, previously associated with aggressive breast cancers, is an endogenous mechanism of non-cycling cells to balance NHEJ (53BP1) and HR (BRCA1). Breast cancer cells exploit this mechanism to ensure genome stability and viability, providing an opportunity for targeted therapy.

C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

Ionizing radiation and genetic risks. XVII. Formation mechanisms underlying naturally occurring DNA deletions in the human genome and their potential relevance for bridging the gap between induced DNA double-strand breaks and deletions in irradiated germ cells

While much is known about radiation-induced DNA double-strand breaks (DSBs) and their repair, the question of how deletions of different sizes arise as a result of the processing of DSBs by the cell's repair systems has not been fully answered. In order to bridge this gap between DSBs and deletions, we critically reviewed published data on mechanisms pertaining to: (a) repair of DNA DSBs (from basic studies in this area); (b) formation of naturally occurring structural variation (SV) - especially of deletions - in the human genome (from genomic studies) and (c) radiation-induced mutations and structural chromosomal aberrations in mammalian somatic cells (from radiation mutagenesis and radiation cytogenetic studies). The specific aim was to assess the relative importance of the postulated mechanisms in generating deletions in the human genome and examine whether empirical data on radiation-induced deletions in mouse germ cells are consistent with predictions of these mechanisms. The mechanisms include (a) NHEJ, a DSB repair process that does not require any homology and which functions in all stages of the cell cycle (and is of particular relevance in G0/G1); (b) MMEJ, also a DSB repair process but which requires microhomology and which presumably functions in all cell cycle stages; (c) NAHR, a recombination-based DSB repair mechanism which operates in prophase I of meiosis in germ cells; (d) MMBIR, a microhomology-mediated, replication-based mechanism which operates in the S phase of the cell cycle, and (e) strand slippage during replication (involved in the origin of small insertions and deletions (INDELs). Our analysis permits the inference that, between them, these five mechanisms can explain nearly all naturally occurring deletions of different sizes identified in the human genome, NAHR and MMBIR being potentially more versatile in this regard. With respect to radiation-induced deletions, the basic studies suggest that those arising as a result of the operation of NHEJ/MMEJ processes, as currently formulated, are expected to be relatively small. However, data on induced mutations in mouse spermatogonial stem cells (irradiation in G0/G1 phase of the cell cycle and DSB repair presumed to be via NHEJ predominantly) show that most are associated with deletions of different sizes, some in the megabase range. There is thus a 'discrepancy' between what the basic studies suggest and the empirical observations in mutagenesis studies. This discrepancy, however, is only an apparent but not a real one. It can be resolved by considering the issue of deletions in the broader context of and in conjunction with the organization of chromatin in chromosomes and nuclear architecture, the conceptual framework for which already exists in studies carried out during the past fifteen years or so. In this paper, we specifically hypothesize that repair of DSBs induced in chromatin loops may offer a basis to explain the induction of deletions of different sizes and suggest an approach to test the hypothesis. We emphasize that the bridging of the gap between induced DSB and resulting deletions of different sizes is critical for current efforts in computational modeling of genetic risks.

Yeast Pol4 Promotes Tel1-Regulated Chromosomal Translocations

DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr540 amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.

Ku70 and non-homologous end joining protect testicular cells from DNA damage.

Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(Δ/Δ) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of γH2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.

Functional analysis—Make or break for cancer predictability

Copy number variations (CNVs) encompass a variety of genetic alterations including deletions and amplifications and cluster in regions of the human genome with intrinsic instability. Small-sized CNVs can act as initial genetic changes giving rise to larger CNVs such as acquired somatic copy number aberrations (CNAs) promoting cancer formation. Previous studies provided evidence for CNVs as an underlying cause of elevated breast cancer risk when targeting breast cancer susceptibility genes and of accelerated breast cancer progression when targeting oncogenes. With the development of novel techniques for genome-wide detection of CNVs at increasingly higher resolution, it became possible to qualitatively and quantitatively analyse manifestation of DNA damage resulting from defects in any of the large variety of DNA double-strand break (DSB) repair mechanisms. Breast carcinogenesis, particularly in familial cases, has been linked with a defect in the homologous recombination (HR) pathway, which in turn switches damage removal towards alternative, more error-prone DSB repair pathways such as microhomology-mediated non-homologous end joining (mmNHEJ). Indeed, increased error-prone DSB repair activities were detected in peripheral blood lymphocytes from individuals with familial breast cancer risk independently of specific gene mutations. Intriguingly, sequence analysis of breakpoint regions revealed that the majority of genome aberrations found in breast cancer specimens are formed by mmNHEJ. Detection of pathway-specific error-prone DSB repair activities by functional testing was proposed to serve as biomarker for hereditary breast cancer risk and responsiveness to therapies targeting HR dysfunction. Identification of specific error-prone DSB repair mechanisms underlying CNAs and ultimately mammary tumour formation highlights potential targets for future breast cancer prevention regimens.

Chromatin modifications and the DNA damage response to ionizing radiation

In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response.

Controlling DNA-end resection: a new task for CDKs

DNA double-strand breaks (DSBs) are repaired by two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). The choice between HR and NHEJ is highly regulated during the cell cycle. DNA-end resection, an evolutionarily conserved process that generates long stretches of single-stranded DNA, plays a critical role in pathway choice, as it commits cells to HR, while, at the same time, suppressing NHEJ. As erroneous DSB repair is a major source of genomic instability-driven tumorigenesis, DNA-end resection factors, and in particular their regulation by post-translational modifications, have become the subject of extensive research over the past few years. Recent work has implicated phosphorylation at S/T-P motifs by cyclin-dependent kinases (CDKs) as a major regulatory mechanism of DSB repair. Intriguingly, CDK activity was found to be critically important for the coordinated and timely execution of DNA-end resection, and key players in this process were subsequently identified as CDK substrates. In this mini review, we provide an overview of the current understanding of how the DNA-end resection machinery in yeast and human cells is controlled by CDK-mediated phosphorylation.

Compromised Nuclear Sirtuins Activity Sensitizes BRCA-Proficient multiple Myeloma Cells to DNA Damage Agents

AbstractAbstract 723 Background:Multiple myeloma (MM) is a clonal malignancy of plasma cells with hallmark genetic instability resulting in large-scale changes at diagnosis, as well as further evolution contributing to disease progression. Inhibition of DNA repair mechanisms leads to significant reduction in acquisition of new genetic changes and associated progression of MM. Mammalian sirtuins are class III NAD+-dependent deacetylases emerging as innovative proteins involved in multiple pathways, including genome maintenance. Methods:A panel of 18 MM cell lines, both sensitive and resistant to conventional and novel anti-MM therapies, was used in the study. The antitumor effect of a pan-sirtuins inhibitor, Nicotinamide (Nam), alone and combined with DNA-damaging agents, was investigated by CTG assay and Annexin-V/propidium iodide staining. Mechanistic studies were performed with thymidine incorporation, Western-blotting, lentivirus-mediated shRNAs and immunofluorescence assay. Analysis of DNA DSB repair was done using chromosomally integrated reporter constructs, followed by cytometer analysis. Results:We analyzed an Affymetrix GeneChip (GSE6477) array of patient MM cells (n=162) compared with normal plasma cells, and found that transcript levels of two nuclear sirtuins (SIRT6 and SIRT7) were significantly higher in monoclonal gammopathy of undetermined significance (MGUS), smoldering MM, active MM, and relapsed MM compared with normal plasma cells. Importantly, protein analysis confirmed increased nuclear levels of these deacetylases in MM cell lines, including those resistant to DNA-damaging agents (MM.1R, LR-5, Dox40), as well as in patient CD138+ MM cells compared to PBMCs from healthy donors. Next we evaluated the functional role of these Sirtuins in MM cells by using loss of function approaches with RNAi. SIRT6 and SIRT7 silencing by knockdown reduced MM cell proliferation compared with control scrambled cells, with only a modest induction of cytotoxicity. We also examined the effects of Nam on DNA-damage response signaling triggered by conventional anti-MM agents melphalan and doxorubicin. Nam treatment did not appreciably affect MM cell viability; however, pretreatment with Nam impaired DNA double-strand breaks (DSBs) repair as well as DNA repair mechanisms triggered by conventional DNA damaging agents, evidenced by γH2AX and RPA phosphorylation, respectively. Consistent with these findings, Nam-pretreated MM cells formed fewer RAD51 foci in response to Doxorubicin and Melphalan, thereby conferring sensitivity to these agents. Importantly, this sensitizing effect was also observed in MM cells resistant to doxorubicin (RPMI-Doxo40) or melphalan (LR5), indicating that Nam increases chemosensitivity in both drug-sensitive and –resistant MM cells. Similarly, lentivirus-mediated shRNA knockdown of SIRT-6 and −7 sensitized MM cells to melphalan and doxorubicin. Finally, both chemical and genetic approaches improved the efficiency of DNA DSB repair mechanisms (Homologous and non-Homologous end-joining Recombination) in MM cell lines containing chromosomally integrated green fluorescent protein-based reporter constructs. Ongoing in vivo experiments are assessing how the chemical susceptibility of SIRT6 and/or 7-deficient cells can be exploited therapeutically. Conclusion:Our study demonstrates a link between nuclear sirtuins and DNA instability in MM cells, providing the basis for incorporation of inhibitors of these SIRTs into innovative anti-MM therapeutic approaches. Disclosures:Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Two Mechanisms Produce Mutation Hotspots at DNA Breaks in Escherichia coli

SummaryMutation hotspots and showers occur across phylogeny and profoundly influence genome evolution, yet the mechanisms that produce hotspots remain obscure. We report that DNA double-strand breaks (DSBs) provoke mutation hotspots via stress-induced mutation in Escherichia coli. With tet reporters placed 2 kb to 2 Mb (half the genome) away from an I-SceI site, RpoS/DinB-dependent mutations occur maximally within the first 2 kb and decrease logarithmically to ∼60 kb. A weak mutation tail extends to 1 Mb. Hotspotting occurs independently of I-site/tet-reporter-pair position in the genome, upstream and downstream in the replication path. RecD, which allows RecBCD DSB-exonuclease activity, is required for strong local but not long-distance hotspotting, indicating that double-strand resection and gap-filling synthesis underlie local hotspotting, and newly illuminating DSB resection in vivo. Hotspotting near DSBs opens the possibility that specific genomic regions could be targeted for mutagenesis, and could also promote concerted evolution (coincident mutations) within genes/gene clusters, an important issue in the evolution of protein functions.

The defect in humoral immunity in patients with Nijmegen breakage syndrome is explained by defects in peripheral B lymphocyte maturation

Patients with an immunodeficiency in the course of Nijmegen breakage syndrome (NBS) that is caused by mutations in the NBN/NBS1 gene are prone to recurrent infections and malignancies, due to a defective DNA double-strand breaks repair mechanism. Four-color flow cytometry was used to analyze changes in B lymphocyte subsets reflecting the most important stages of peripheral B cell maturation. It was demonstrated that the humoral immune defect observed in NBS patients was caused by reduced numbers of B lymphocytes, but also by their aberrant maturation. Reduced relative and absolute counts of naïve and memory B cells were accompanied by a significant accumulation of the natural effector B lymphocytes. The elevated proportion of IgM-only memory and reduced proportion of IgM-negative cells within the memory B cell pool suggests that there is class-switch recombination defect in this population of cells in NBS patients, resulting in inadequate production of immunoglobulins. Because of the reduced T-cell counts, the T-cell dependent antigen response is severely impaired resulting in a lower frequency of memory B-cells. The T-cell independent B-cell differentiation pathway seems less affected. The reduced IgG and IgA levels in patients with NBS are caused both by ineffective class switch, at least due to poor T cell help, and low number of memory B cells. This study illustrates that the NBN gene product nibrin plays an important role at different levels in the B-cell system.

Analysis and results of Ku and XRCC4 expression in hypopharyngeal cancer tissues treated with chemoradiotherapy

DNA double-strand break (DSB) is one of the most serious forms of damage induced by ionizing irradiation. Non-homologous end-joining (NHEJ) is a key mechanism of DNA DSB repair. The immunohistochemical analysis of proteins involved in NHEJ may have potential as a predictive assay for tumor radiosensitivity. We examined the correlation between the expression of proteins involved in DNA DSB in biopsy specimens and the results of chemoradiotherapy in hypopharyngeal cancers. Fifty-seven patients with previously untreated squamous cell carcinoma of the hypopharynx were treated between March 2002 and December 2009. Most patients (75%) had stage III or IV disease. The chemotherapy consisted of cisplatin plus 5FU or S-1. A tumor dose of 50 Gy was usually administered to the primary tumor and regional lymph nodes. Doses of 10-20 Gy were usually added to the primary tumor with reduced fields after 50 Gy. The 5-year disease-free survival rate was 100% for patients in stage I, 90% in stage II, 64% in stage III and 50% in stage IV. In stages I-III, patients with a lower expression of Ku70 or XRCC4 tended to have better locoregional control. These results indicated that a lower expression of Ku70 or XRCC4 may be correlated with higher radiosensitivity. Two patients had distant metastasis alone, of which one had 0% expression of Ku70 and the other had 0% expression of Ku86. The absence of Ku70 or Ku86 expression indicates low DNA-PK activity. Low DNA-PK activity due to a low expression of Ku may result in the genetic alteration of cancer cells, leading to a higher tendency of distant metastasis. This finding suggests that proteins involved in NHEJ may have an impact on the treatment results of chemoradiotherapy in hypopharyngeal cancer.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development.

Repairing breaks in the plant genome: the importance of keeping it together

DNA damage threatens the integrity of the genome and has potentially lethal consequences for the organism. Plant DNA is under continuous assault from endogenous and environmental factors and effective detection and repair of DNA damage are essential to ensure the stability of the genome. One of the most cytotoxic forms of DNA damage are DNA double-strand breaks (DSBs) which fragment chromosomes. Failure to repair DSBs results in loss of large amounts of genetic information which, following cell division, severely compromises daughter cells that receive fragmented chromosomes. This review will survey recent advances in our understanding of plant responses to chromosomal breaks, including the sources of DNA damage, the detection and signalling of DSBs, mechanisms of DSB repair, the role of chromatin structure in repair, DNA damage signalling and the link between plant recombination pathways and transgene integration. These mechanisms are of critical importance for maintenance of plant genome stability and integrity under stress conditions and provide potential targets for the improvement of crop plants both for stress resistance and for increased precision in the generation of genetically improved varieties.

Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

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.

Evaluation of DNA Double Strand Breaks Repair Efficiency in Head and Neck Cancer

Head and neck cancers (head and neck squamous cell carcinomas [HNSCC]) are a heterogeneous group of neoplasms with varying presenting symptoms, treatment, and expected outcome. There is a need to find an effective way of its treatment at the molecular level. Thus, we should identify the mechanism of cancer cell response to damaging agents' activity, especially at DNA level. Our major goal was to evaluate the efficacy of DNA double strand breaks (DSBs) repair in HTB-43 and SCC-25 cancer cell lines as well as lymphocytes taken from HNSCC patients and healthy donors. The DNA repair efficiency was measured by neutral comet assay as well as extrachromosomal assay for DNA DSBs repair (TAK assay). We determined the levels of two main pathways of DNA DSBs-nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). Neutral comet assay was used for evaluation of DNA DSBs repair after treatment with genotoxic agents. DNA DSBs induced by gamma radiation were repaired slower in lymphocytes from HNSCC patients than in lymphocytes from healthy controls. HTB-43 and SCC-25 cancer cell lines have higher efficacy of NHEJ and HRR than lymphocytes taken from patients as well as control subjects. Our results confirm the necessity of further studies on the mechanisms of DNA DSBs repair to provide insight into the molecular basis of head and neck cancer, which will allow us to improve methods of HNSCC treatment.

Double-strand breaks repair by gene conversion in silkworm holocentric chromosomes

Maintenance of genome stability relies on the accurate repair of DNA double-strand breaks (DSBs) that arise during DNA replication or introduced by DNA-damaging agents. Failure to repair such breaks can lead to the introduction of mutations and chromosomal translocations. Several pathways, homologous recombination, single-strand annealing and nonhomologous end-joining, are known to repair DSBs. So far in the silkworm Bombyx mori, these repair pathways have been analyzed using extrachromosomal plasmids in vitro or in cultured cells. To elucidate the precise nature of the chromosomal DSB repair pathways in cultured silkworm cells, we developed a luciferase-based assay system for measuring the frequency of chromosomal homologous recombination and SSA. An I-SceI-induced DSB, within a nonfunctional luciferase gene, could be efficiently repaired by HR. Additionally, the continuous expression of the I-SceI endonuclease in the HR reporter cell allowed us to investigate the interrelationship between HR, SSA and NHEJ. In this study, we demonstrated that chromosome DSBs were mainly repaired by NHEJ and HR, whereas SSA was unlikely to be a dominant repair pathway in cultured silkworm cell. These results indicate that the assay system presented here will be useful to analyze the mechanisms of DSB repair in insect cells.

Screening from the world's largest TCM database for inhibiting DNA repair protein XRCC4

Human XRCC4 protein is a key component of DNA double-strand break (DSB) repair pathway related to the diseases of stroke and cancer. Cancer cells being treated with the drugs that interfere with DSBs repair mechanism have shown increased radiosensitivity to ionising radiation. Therefore, the development of novel radiosensitiser for radiation therapy becomes important for cancer treatment. We screened from the world's largest traditional Chinese medicine (TCM) database and found potential TCM molecules that can dock at XRCC4 functional site. Among the selected potential TCM compounds, we specifically investigated the top-ranked molecules: glycyrrhizic acid and macedonoside C. The molecular docking and molecular dynamics simulations on these compounds show similar location with high predicted binding affinity. Both compounds form continuous interaction with Lys188 and Arg192 of chain C and Lys187 and Lys190 of chain D. All these protein residues are required to form key hydrophobic interactions with other components participating in DNA repair. We suggested both glycyrrhizic acid and macedonoside C as potential lead compounds for inhibiting XRCC4.

The BRCA1-RAP80 Complex Regulates DNA Repair Mechanism Utilization by Restricting End Resection

The tumor suppressor protein BRCA1 is a constituent of several different protein complexes and is required for homology-directed repair (HDR) of DNA double strand breaks (DSBs). The most recently discovered BRCA1-RAP80 complex is recruited to ubiquitin structures on chromatin surrounding the break. Deficiency of any member of this complex confers hypersensitivity to DNA-damaging agents by undefined mechanisms. In striking contrast to other BRCA1-containing complexes that are known to promote HDR, we demonstrate that the BRCA1-RAP80 complex restricts end resection in S/G(2) phase of the cell cycle, thereby limiting HDR. RAP80 or BRCC36 deficiency resulted in elevated Mre11-CtIP-dependent 5' end resection with a concomitant increase in HDR mechanisms that rely on 3' single-stranded overhangs. We propose a model in which the BRCA1-RAP80 complex limits nuclease accessibility to DSBs, thus preventing excessive end resection and potentially deleterious homology-directed DSB repair mechanisms that can impair genome integrity.