Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.     

Inhibition of DNA damage repair by artificial activation of PARP with siDNA

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.     

The role of NBS1 in DNA double strand break repair, telomere stability, and cell cycle checkpoint control

The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS 1 complex （MRN） that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome （NBS）, a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance oftelomere stability.     

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

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.     

NBS1在DNA断裂损伤反应和维持端粒稳定中的作用

NBS1作为MRE11/RAD50/NBS1复合物的组分之一,是细胞应答DNA损伤的一个关键蛋白质,在DNA双链断裂修复和维持基因组稳定中发挥重要的作用。端粒是染色体末端由DNA重复序列和蛋白质构成的复合体,其独特结构与DNA双链断裂非常相似。最近几年的研究发现NBS1与端粒也有着十分密切的联系。综述了NBS1在DNA损伤反应中的作用,并探讨NBS1参与维持端粒稳定中的分子机制。     

DNA repair factors and telomere-chromosome integrity in mammalian cells

Loss of telomere equilibrium and associated chromosome-genomic instability might effectively promote tumour progression. Telomere function may have contrasting roles: inducing replicative senescence and promoting tumourigenesis and these roles may vary between cell types depending on the expression of the enzyme telomerase, the level of mutations induced, and efficiency/deficiency of related DNA repair pathways. We have identified an alternative telomere maintenance mechanism in mouse embryonic stem cells lacking telomerase RNA unit (mTER) with amplification of non-telomeric sequences adjacent to existing short stretches of telomere repeats. Our quest for identifying telomerase-independent or alternative mechanisms involved in telomere maintenance in mammalian cells has implicated the involvement of potential DNA repair factors in such pathways. We have reported earlier on the telomere equilibrium in scid mouse cells which suggested a potential role of DNA repair proteins in telomere maintenance in mammalian cells. Subsequently, studies by us and others have shown the association between the DNA repair factors and telomere function. Mice deficient in a DNA-break sensing molecule, PARP-1 (poly [ADP]-ribopolymerase), have increased levels of chromosomal instability associated with extensive telomere shortening. Ku80 null cells showed a telomere shortening associated with extensive chromosome end fusions, whereas Ku80+/- cells exhibited an intermediate level of telomere shortening. Inactivation of PARP-1 in p53-/- cells resulted in dysfunctional telomeres and severe chromosome instability leading to advanced onset and increased tumour incidence in mice. Interestingly, haploinsufficiency of PARP-1 in Ku80 null cells causes more severe telomere shortening and chromosome abnormalities compared to either PARP-1 or Ku80 single null cells and Ku80+/-PARP-/- mice develop spontaneous tumours. This overview will focus mainly on the role of DNA repair/recombination and DNA damage signalling molecules such as PARP-1, DNA-PKcs, Ku70/80, XRCC4 and ATM which we have been studying for the last few years. Because the maintenance of telomere function is crucial for genomic stability, our results will provide new insights into the mechanisms of chromosome instability and tumour formation.     

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.     

DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells

NBS1-deficient cells exhibit pronounced radiosensitivity and defects in chromosome integrity after ionizing radiation (IR) exposure, yet show only a minor defect in DNA double-strand break (DSB) rejoining, leaving an as yet unresolved enigma as to the nature of the radiosensitivity of these cells. To further investigate the relationship between radiosensitivity, DSB repair, and chromosome stability, we have compared cytological and molecular assays of DSB misrejoining and repair in NBS1-defective, wild type, and NBS1-complemented cells after IR damage. Our findings suggest a subtle defect in overall DSB rejoining in NBS1-defective cells and uniquely also reveal reduced ability of NBS1-defective cells to rejoin correct ends of DSBs. In agreement with published results, one of two different NBS1-defective cell lines showed a slight defect in overall rejoining of DSBs compared to its complemented counterpart, whereas another NBS line did not show any difference from wild type cells. Significant defects in the correct rejoining of DSBs compared to their respective controls were observed for both NBS1-defective lines. The defect in DSB rejoining and the increased misrejoining detected at the molecular level were also reflected in higher levels of fragments and translocations, respectively, at the chromosomal level. This work provides both molecular and cytological evidence that NBS1-deficient cells have defects in DSB processing and reveals that these molecular events can be manifest cytologically.     

Hepatitis C virus infection activates the immunologic (type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes

Hepatitis C virus (HCV) infection causes hepatitis, hepatocellular carcinoma, and B-cell lymphomas in a significant number of patients. Previously we have shown that HCV infection causes double-stranded DNA breaks and enhances the mutation frequency of cellular genes, including proto-oncogenes and immunoglobulin genes. To determine the mechanisms, we studied in vitro HCV infection of cell culture. Here we report that HCV infection activated the immunologic (type II) isoform of nitric oxide (NO) synthase (NOS), i.e., inducible NOS (iNOS), thereby inducing NO, which in turn induced DNA breaks and enhanced the mutation frequencies of cellular genes. Treatment of HCV-infected cells with NOS inhibitors or small interfering RNA specific for iNOS abolished most of these effects. Expression of the core protein or nonstructural protein 3 (NS3), but not the other viral proteins, in B cells or hepatocytes induced iNOS and DNA breaks, which could be blocked by NOS inhibitors. The core protein also enhanced the mutation frequency of cellular genes in hepatocytes derived from HCV core transgenic mice compared with that in control mice. The iNOS promoter was activated more than fivefold in HCV-infected cells, as revealed by a luciferase reporter assay driven by the iNOS promoter. Similarly, the core and NS3 proteins also induced the same effects. Therefore, we conclude that HCV infection can stimulate the production of NO through activation of the gene for iNOS by the viral core and NS3 proteins. NO causes DNA breaks and enhances DNA mutation. This sequence of events provides a mechanism for HCV pathogenesis and oncogenesis.     

Role of RAD51C and XRCC3 in genetic recombination and DNA repair

In germ line cells, recombination is required for gene reassortment and proper chromosome segregation at meiosis, whereas in somatic cells it provides an important mechanism for the repair of DNA double-strand breaks. Five proteins (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) that share homology with RAD51 recombinase and are known as the RAD51 paralogs are important for recombinational repair, as paralog-defective cell lines exhibit spontaneous chromosomal aberrations, defective DNA repair, and reduced gene targeting. The paralogs form two distinct protein complexes, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3, but their precise cellular roles remain unknown. Here, we show that, like MLH1, RAD51C localized to mouse meiotic chromosomes at pachytene/diplotene. Using immunoprecipitation and gel filtration analyses, we found that Holliday junction resolvase activity associated tightly and co-eluted with the 80-kDa RAD51C-XRCC3 complex. Taken together, these data indicate that the RAD51C-XRCC3-associated Holliday junction resolvase complex associates with crossovers and may play an essential role in the resolution of recombination intermediates prior to chromosome segregation.     

Roles of nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage

Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.     

DNA repair and chromosomal alterations

All mutagenic agents induce lesions in the cellular DNA and they are repaired efficiently by different repair mechanisms. Un-repaired and mis-repaired lesions lead to chromosomal aberrations (CAs). Depending upon the mutagenic agents involved, different DNA repair pathways, such as nucleotide excision repair (NER), base excision repair (BER), non-homologous end joining (NHEJ), homologous recombination repair (HRR), cross-link repair (FANC), single strand annealing (SSA) etc., are operative. Following ionising radiation, DNA double strand breaks (DSBs, which are considered to be the most important leasion leading to observed biological effects) are repaired either by NHEJ and/or HRR. We have investigated the relative role of these two repair pathways leading to chromosomal aberrations using Chinese hamster ovary (CHO) mutant cells deficient in one of these two repair pathwatys. NHEJ operates both in G1 and G2 phases of the cell cycle, wheras HHR operates mainly in S and G2 phases of the cell cycle. In NHEJ-deficient mutant cells irradiated in G1, un-repaired double strand breaks reaching S phase are repaired (unexpectedly with a large mis-repair component) by HRR. In HRR-deficient mutant cells, un-repaired DSBs reaching S phase are repaired by NHEJ (unexpectedly with a low mis-repair component) as evidenced by the frequencies of chromatid type aberrations. Employing a similar approach, following treatment with benzo(alpha)pyrene-7,8diol-9,10epoxide (BPDE), the active metabolite of benzo(alpha)pyrene, NER and HRR seem to be the most important repair pathways protecting against chromosomal damage induced by this agent. In the case of acetaldehyde, (primary metabolite of alcohol in vivo) a DNA cross-linking agent, HRR and FANC pathways are important for protection against damage induced by this agent. Irrespective of the type of DNA lesions induced, ultimately they have to be converted to DSBs in order to give rise to CA. Therefore, both NHEJ and HRR are also involved to some extent in the origin of CA following treatment with S-dependent agents.The relative importance of different repair pathways in bestowing protection against DNA damage leading to chromosomal alterations is discussed.     

Chromosome segregation and double-strand break repair - a complex connection

Genome stability requires correct chromosome segregation and DNA repair. Failure of these processes leads to cell death or accumulation of chromosomal aberrations, as often observed in tumor cells. An increasing number of observations indicate that segregation and DNA double-strand break (DSB) repair are functionally connected by the Cohesin and Smc5/6 protein complexes. Through their interaction with the duplicated genome, these complexes play essential roles in both chromosome segregation and repair by sister chromatid recombination. Both are also recruited to DSBs, and their chromosomal association is similarly regulated. Interestingly, recent studies of Cohesin suggest that DSB formation could promote proper mitotic chromosome segregation. This is reminiscent of segregation in meiotic cells, which is facilitated by break-induced chromosomal tethering.     

NBS1 is involved in DNA repair and plays a synergistic role with ATM in mediating meiotic homologous recombination in plants

The ability of plants to repair DNA double-strand breaks (DSBs) is essential for growth and fertility. The Arabidopsis DSB repair proteins AtRAD50 and AtMRE11 form part of an evolutionarily conserved complex that, in Saccharomyces cerevisiae and mammals, includes a third component termed XRS2 and NBS1, respectively. The MRN complex (MRX in yeast) has a direct role in DSB repair and is also required for DNA damage signaling and checkpoint activation in a pathway mediated by the protein kinase ATM. This study characterizes Arabidopsis and maize NBS1 orthologues that share conserved protein motifs with human NBS1. Both plant NBS1 proteins interact with the corresponding MRE11 orthologues, and deletion analysis of AtNBS1 defines a region towards the C-terminus (amino acids 465-500) that is required for interaction with AtMRE11. Arabidopsis lines homozygous for a T-DNA insertional mutation in AtNBS1 display hypersensitivity to the DNA cross-linking reagent mitomycin C, and this phenotype can be rescued by complementation with the wild-type gene, consistent with a function for AtNBS1 in plant DSB repair. Analysis of atnbs1-1 atatm double mutants revealed a role for AtNBS1 in meiotic recombination. While atatm mutants produce reduced seed numbers, plants deficient in both AtATM and AtNBS1 are completely infertile. Cytological analysis of these double mutants revealed incomplete chromosome pairing and synapsis in meiotic prophase, and extensive chromosome fragmentation in metaphase I and subsequent stages. These results suggest a novel role for AtNBS1 that is independent of AtATM-mediated signaling and functions in the very early stages of meiosis.     

The Nijmegen breakage syndrome protein is essential for Mre11 phosphorylation upon DNA damage.

The Nijmegen breakage syndrome (NBS), a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. Repair of DNA double-strand breaks by radiation is dependent on a multifunctional complex containing Rad50, Mre11, and the NBS1 gene product, p95 (NBS protein, nibrin). The role of p95 in these repair processes is unknown. Here it is demonstrated that Mre11 is hyperphosphorylated in a cell cycle-independent manner in response to treatment of cells with genotoxic agents including gamma irradiation. This response is abrogated in two independently established NBS cell lines that have undetectable levels of the p95 protein. NBS cells are also deficient for radiation-induced nuclear foci containing Mre11, while those with Rad51 are unaffected. An analysis of the kinetic relationship between Mre11 phosphorylation and the appearance of its radiation-induced foci indicates that the former precedes the latter. Together, these data suggest that specific phosphorylation of Mre11 is induced by DNA damage, and p95 is essential in this process, perhaps by recruiting specific kinases.     

Targeted disruption of NBS1 reveals its roles in mouse development and DNA repair

Nijmegen breakage syndrome (NBS) is an autosomal recessive hereditary disease that shares some common defects with ataxia-telangiectasia. The gene product mutated in NBS, named NBS1, is a component of the Mre11 complex that is involved in DNA strand-break repair. To elucidate the physiological roles of NBS1, we disrupted the N-terminal exons of the NBS1 gene in mice. NBS1(m/m) mice are viable, growth retarded and hypersensitive to ionizing radiation (IR). NBS1(m/m) mice exhibit multiple lymphoid developmental defects, and rapidly develop thymic lymphoma. In addition, female NBS1(m/m) mice are sterile due to oogenesis failure. NBS1(m/m) cells are impaired in cellular responses to IR and defective in cellular proliferation. Most systematic and cellular defects identified in NBS1(m/m) mice recapitulate those in NBS patients, and are essentially identical to those observed in Atm(-/-) mice. In contrast to Atm(-/-) mice, spermatogenesis is normal in NBS1(m/m) mice, indicating that distinct roles of ATM have differential requirement for NBS1 activity. Thus, NBS1 and ATM have overlapping and distinct functions in animal development and DNA repair.