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PubMed1995

Menage à trois: double strand break repair, V(D)J recombination and DNA-PK.

All organisms possess mechanisms to repair double strand breaks (dsbs) generated in their DNA by damaging agents. Site-specific dsbs are also introduced during V(D)J recombination. Four complementation groups of radiosensitive rodent mutants are defective in the repair of dsbs, and are unable to carry out V(D)J recombination effectively. The immune defect in Severe Combined Immunodeficient (scid) mice also results from an inability to undergo effective V(D)J recombination, and scid cell lines display a repair defect and belong to one of these complementation groups. These findings indicate a mechanistic overlap between the processes of DNA repair and V(D)J recombination. Recently, two of the genes defined by these complementation groups have been identified and shown to encode components of DNA-dependent protein kinase (DNA-PK). We review here the three fields which have become linked by these findings, and discuss the involvement of DNA-PK in dsb rejoining and in V(D)J recombination.

Animals↗

PubMed2005

BCR/ABL modifies the kinetics and fidelity of DNA double-strand breaks repair in hematopoietic cells.

The oncogenic BCR/ABL tyrosine kinase facilitates the repair of DNA double-strand breaks (DSBs). We find that after gamma-irradiation BCR/ABL-positive leukemia cells accumulate more DSBs in comparison to normal cells. These lesions are efficiently repaired in a time-dependent fashion by BCR/ABL-stimulated non-homologous end-joining (NHEJ) followed by homologous recombination repair (HRR) mechanisms. However, mutations and large deletions were detected in HRR and NHEJ products, respectively, in BCR/ABL-positive leukemia cells. We propose that unfaithful repair of DSBs may contribute to genomic instability in the Philadelphia chromosome-positive leukemias.

Animals↗

PubMed2004

Incidence of Fas positivity and deoxyribonucleic acid double-stranded breaks in human ejaculated sperm.

OBJECTIVE: To determine the incidence of Fas positivity and DNA double-strand breaks (DSB) as indicators of early- and late-stage apoptosis in ejaculated sperm. DESIGN: Fas positivity was assessed by flow cytometry and DSB by the neutral Comet assay. SETTING: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. PATIENT(S) AND INTERVENTION(S): Forty-five infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies. MAIN OUTCOME MEASURE(S): Percentage Fas-positive cells, percentage DNA fragmentation, olive tail moment. RESULT(S): The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozospermic semen. No Fas positivity was observed in fertile men's sperm. Deoxyribonucleic acid fragmentation (DSB) was greater in infertile than in fertile men's sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage. CONCLUSION(S): Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis.

Apoptosis↗

PubMed2006

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks.

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.

Amino Acids, Cyclic↗

PubMed1987

Effect of cellular glutathione content on the induction of DNA double strand breaks by 25 MeV electrons.

The effect of endogenous glutathione (GSH) on the induction of DNA double strand breaks (dsb) by 25 MeV electrons was investigated using stationary haploid yeast cells defective in gamma-glutamyl-cysteine-synthetase (gsh 1) containing less than 5 per cent of the normal GSH content. In gsh 1 cells the induction of dsb is increased by a factor of 1.5 under oxic and 1.8 under anoxic irradiation conditions: whereas the oxygen enhancement ratio was only slightly decreased (1.9) compared to wild-type cells (2.4).

Cell Survival↗

PubMed1993

Influence of irradiation conditions on the measurement of DNA double-strand breaks by pulsed-field gel electrophoresis.

The induction of gamma-ray-induced DNA double-strand breaks (DSBs) determined by pulsed-field gel electrophoresis was compared in the DNA of intact Chinese hamster ovary (CHO) cells imbedded in agarose plugs, and in the isolated DNA of agarose-imbedded CHO cells that had been lysed before irradiation, to determine whether these irradiation conditions would influence their measurement. DSB-induction in irradiated cells or isolated DNA was measured as the loss of DNA from the plug compared with the unirradiated control. Chromatin protein was completely digested by the lysis buffer and as such did not affect DNA migration upon electrophoresis, whereas concentrations of EDTA as low as 10(-5) M affected the induction of DSBs in the isolated DNA. The gel plugs required several hours of washing with PBS to remove the contaminating EDTA from the lysis buffer. Once the residual EDTA was removed, DNA DSB induction as a function of dose was 70 times greater in isolated DNA than in the DNA of intact cells.

Animals↗

PubMed2003

Polymorphisms in DNA double-strand break repair genes and breast cancer risk in the Nurses' Health Study.

Genetic polymorphisms in double-strand break repair genes may influence DNA repair capacity and, in turn, confer predisposition to breast cancer. We prospectively assessed the associations of candidate polymorphisms G31479A (R188H) in XRCC2, A4541G (5'-UTR), A17893G (IVS5-14) and C18067T (T241 M) in XRCC3, and C299T (5'-UTR) and T1977C (D501D) in Ligase IV with breast cancer risk in a nested case-control study within the Nurses' Health Study (incident cases, n=1004; controls, n=1385). We observed no overall associations of these six genotypes with breast cancer risk. Four common haplotypes in XRCC3 accounted for 99% of the chromosomes of the present study population. We observed that Ligase IV 1977C carriers were at increased breast cancer risk if they had a first degree family history of breast cancer (test for interaction, P=0.01). We observed that the XRCC2 R188H polymorphism modified the association of plasma alpha-carotene level and breast cancer risk (test for ordinal interaction, P=0.03); the significantly decreased risk seen overall for women in the highest quartile of plasma alpha-carotene was only present among 188H non-carriers (the top quartile versus the bottom quartile; multivariate odds ratio, 0.55; 95% confidence interval, 0.40-0.75). No significant interactions were seen between any of these polymorphisms and duration or dose of cigarette smoking. The gene-environment interaction data suggest that the subtle effects of some of these variants on breast cancer risk may be magnified in the presence of certain exposures.

Adult↗

PubMed1999

Mechanisms of double-strand-break repair during gene targeting in mammalian cells.

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting. In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp. The hDNA was efficiently repaired prior to DNA replication. The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.

Animals↗

PubMed1997

Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.

Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways. The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells. Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs. The addition of Cre recombinase activity promotes deletions of three types that can be scored. We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele. However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed. Therefore, Mre11 is required for normal cell proliferation. This methodology introduces a means to study important regions of essential genes in cell culture models.

Amino Acid Sequence↗

PubMed1998

Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells.

To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.

Amino Acid Sequence↗

PubMed2000

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

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.

Animals↗

PubMed2006

Saccharomyces cerevisiae Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation.

In budding yeast, at least 10 proteins are required for formation of the double-strand breaks (DSBs) that initiate meiotic recombination. Spo11 is the enzyme responsible for cleaving DNA and is found in a complex that also contains Ski8, Rec102, and Rec104. The Mre11/Rad50/Xrs2 complex is required for both DSB formation and DSB processing. In this article we investigate the functions of the remaining three proteins--Mer2, Mei4, and Rec114--with particular emphasis on Mer2. The Mer2 protein is present in vegetative cells, but it increases in abundance and becomes phosphorylated specifically during meiotic prophase. Mer2 localizes to distinct foci on meiotic chromosomes, with foci maximally abundant prior to the formation of synaptonemal complex. If DSB formation is blocked (e.g., by a spo11 mutation), dephosphorylation of Mer2 and its dissociation from chromosomes are delayed. We have also found that the Mei4 and Rec114 proteins localize to foci on chromosomes and these foci partially colocalize with each other and with Mer2. Furthermore, the three proteins co-immunoprecipitate. Mer2 does not show significant colocalization with Mre11 or Rec102 and Mer2 does not co-immunoprecipitate with Rec102. We propose that Mer2, Mei4, and Rec114 form a distinct complex required for DSB formation.

Chromosome Breakage↗

PubMed2004

Role of the nuclease activity of Saccharomyces cerevisiae Mre11 in repair of DNA double-strand breaks in mitotic cells.

The Rad50:Mre11:Xrs2 (RMX) complex functions in repair of DNA double-strand breaks (DSBs) by recombination and nonhomologous end-joining (NHEJ) and is also required for telomere stability. The Mre11 subunit exhibits nuclease activities in vitro, but the role of these activities in repair in mitotic cells has not been established. In this study we have performed a comparative study of three mutants (mre11-D16A, -D56N, and -H125N) previously shown to have reduced nuclease activities in vitro. In ends-in and ends-out chromosome recombination assays using defined plasmid and oligonucleotide DNA substrates, mre11-D16A cells were as deficient as mre11 null strains, but defects were small in mre11-D56N and -H125N mutants. mre11-D16A cells, but not the other mutants, also displayed strong sensitivity to ionizing radiation, with residual resistance largely dependent on the presence of the partially redundant nuclease Exo1. mre11-D16A mutants were also most sensitive to the S-phase-dependent clastogens hydroxyurea and methyl methanesulfonate but, as previously observed for D56N and H125N mutants, were not defective in NHEJ. Importantly, the affinity of purified Mre11-D16A protein for Rad50 and Xrs2 was indistinguishable from wild type and the mutant protein formed complexes with equivalent stoichiometry. Although the role of the nuclease activity has been questioned in previous studies, the comparative data presented here suggest that the nuclease function of Mre11 is required for RMX-mediated recombinational repair and telomere stabilization in mitotic cells.

DNA↗

PubMed2001

Heavy ion production of single- and double-strand breaks in plasmid DNA in aqueous solution.

Preliminary measurements on the production of single- and double-strand breaks in SV40 and phi X174 plasmid DNA by various heavy ions in radioprotective Tris buffer are presented. The dependence of the recorded yields on the LET of the incoming ions is discussed and shown to be comprehensible within the framework of a model based on the X-ray sensitivity of the exposed DNA and the local dose distribution inside the heavy ion track. The question of the influence of the chemical environment is addressed by comparing the measured cross sections to data recorded previously in more radiosensitive TE buffer. The results indicate that also for systems with high scavenging capacity RBE values larger than unity can be achieved with maximum values in the LET range 100-1000 keV micrometers-1.

Bacteriophage phi X 174↗

PubMed2002

Misrepair of radiation-induced DNA double-strand breaks and its relevance for tumorigenesis and cancer treatment (review).

The faithful repair of DNA double-strand breaks (DSBs) is probably one of the most critical tasks for a cell in order to maintain its genomic integrity since these lesions may lead to chromosome breaks or rearrangements, mutations, cell death or cancer. DSBs can arise spontaneously during normal cellular DNA metabolism or may be induced by exogenous agents such as ionizing radiation. To overcome the danger that emanates from these lesions, eukaryotic cells have evolved specific pathways for processing DSBs by either homology-dependent or non-homologous repair mechanisms. This review focuses on the formation of genomic rearrangements that arise by joining incorrect break ends and on the factors that influence repair fidelity. Recent studies indicate that the probability for a break to be incorrectly rejoined is fairly low when DSBs are spatially separated but increases drastically when multiple breaks coincide. The formation of genomic rearrangements in situations of multiple breaks is mediated by non-homologous end-joining, the predominant DSB repair pathway in mammalian cells. Interestingly, the same pathway is required for efficiently preserving chromosomal integrity in situations of separated breaks. Furthermore, the probability for a DSB to be faithfully repaired depends on its genomic location and on the cell cycle position. Methods for assaying DSB repair are discussed, again with emphasis on experimental systems that allow to determine whether a DSB is correctly or incorrectly rejoined.

Animals↗

PubMed2003

[Construction of double-strand break repair protein hKu70 deficient cell strain and its biologic characters].

OBJECTIVE: To construct DNA double-strand break (DSB) repair protein hKu70 deficient cell strain and to observe its biological characters for studying the functions of hKu70 gene and the effects of occupational harmfulness factors on DSB repair. METHODS: Human lung fibroblasts (HLF) were transfected with the eukaryotic expression plasmids of hKu70 gene antisense RNA (pEGFP-C1-K) to construct hKu70 protein deficient cells (named as "HLFK"). The protein expression levels of hKu70 gene in HLFC and HLFK were detected by the Western blotting to estimate the effects of antisense inhibition. Morphology, growth character and growth status in soft agar of transfected HLFK were observed. RESULTS: pEGFP-C1-K vector was successfully expressed in HLF. The protein expression level of hKu70 gene in HLFK was decreased by 42% as compared with that in HLFC. No obvious changes of the biologic characters were observed in HLFK. CONCLUSION: The hKu70 protein deficient cell strain was successfully constructed. The hKu70 protein deficiency alone didn't induce obvious changes of the biological characters in HLFK.

Antigens, Nuclear↗

PubMed1984

Repair of DNA double-strand breaks as a determinant of RBE of alpha particles.

The role of repair of DNA double-strand breaks (dsb) in the determination of the RBE-value of alpha particles was studied using the temperature conditional radiosensitive diploid yeast mutant rad 54-3. This mutant is proficient in the repair of dsb at the permissive temperature of 23 degrees C at which it yields a shouldered survival curve, but it is dsb repair-deficient at the restrictive temperature of 36 degrees C at which it yields an exponential survival curve. At the permissive temperature the rad 54-3 mutant also shows liquid holding recovery of colony forming ability as a function of the liquid holding period. Thus, with this mutant it is possible to obtain survival curves involving no repair of dsb (immediate plating, 36 degrees C), partial repair of dsb (immediate plating, 23 degrees C) and gradually increasing levels of dsb repair by delayed plating after liquid holding periods of 24, 48 and 72 h. The RBE-values of densely ionizing 3.5 MeV alpha particles for cell killing relative to sparsely ionizing 30 MeV electrons have been determined as a function of the level of dsb repair. It is shown that the RBE-value is low and independent of dose when no repair of dsb is involved, whereas it becomes gradually larger with a gradual increase in the level of dsb repair.

Alpha Particles↗

PubMed2005

Mdm2 binds to Nbs1 at sites of DNA damage and regulates double strand break repair.

Mdm2 directly regulates the p53 tumor suppressor. However, Mdm2 also has p53-independent activities, and the pathways that mediate these functions are unresolved. Here we report the identification of a specific association of Mdm2 with Mre11, Nbs1, and Rad50, a DNA double strand break repair complex. Mdm2 bound to the Mre11-Nbs1-Rad50 complex in primary cells and in cells containing inactivated p53 or p14/p19ARF, a regulator of Mdm2. Further analysis revealed that Mdm2 directly bound to Nbs1 but not to Mre11 or Rad50. Amino acids 198-314 of Mdm2 were required for Mdm2/Nbs1 association, and neither the N terminus forkhead-associated and breast cancer C-terminal domains nor the C terminus Mre11 binding domain of Nbs1 mediated the interaction of Nbs1 with Mdm2. Mdm2 co-localized with Nbs1 to sites of DNA damage following gamma-irradiation. Notably, Mdm2 overexpression inhibited DNA double strand break repair, and this was independent of p53 and ARF, the alternative reading frame of the Ink4alocus. The delay in DNA repair imposed by Mdm2 required the Nbs1 binding domain of Mdm2, but the ubiquitin ligase domain in Mdm2 was dispensable. Therefore, Nbs1 is a novel p53-independent Mdm2 binding protein and links Mdm2 to the Mre11-Nbs1-Rad50-regulated DNA repair response.

Acid Anhydride Hydrolases↗