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PubMed1993

Arsenite enhances DNA double-strand breaks and cell killing of methyl methanesulfonate-treated cells by inhibiting the excision of alkali-labile sites.

Analysis of DNA strand breaks by alkaline elution indicates that DNA repair of Chinese hamster ovary cells treated with methyl methanesulfonate (MMS) was inhibited by sodium arsenite. Comparing the profiles of a 36-min elution with buffer pH 12.1 and a 12-h elution with buffer pH 12.4 revealed that alkali-labile sites were increased more than frank breaks in the combined treatment with MMS plus arsenite. Enhancement of alkali-labile sites was detected with low doses of MMS and arsenite, whereas enhancement of frank breaks required higher doses of MMS and arsenite. Double-strand breaks were detected after incubating the MMS-treated cells in an arsenite-containing medium for 18 or 12 h but not less than 6 h. No double-strand breaks were detected when MMS-damaged cells were posttreated with arsenite for 3 h; however, double-strand breaks were detected after further incubating these cells in arsenite-free medium for 18 h. Thus, inhibition of arsenite on the excision of methylated bases may have accumulated a large number of alkali-labile sites in the parental strands, and DNA replication may then generate breaks in the non-methylated daughter strands. Double-strand breaks may result from overlapping gaps between the parental and daughter strands and/or postreplication repair. These double-strand breaks may then result in the synergistic cell death as observed with posttreatment of MMS-damaged cells with arsenite for 1 or 3 h.

Alkylating Agents↗

PubMed2003

Relationship of DNA double-strand breaks to synapsis in Drosophila.

The relationship between synaptonemal complex formation (synapsis) and double-strand break formation (recombination initiation) differs between organisms. Although double-strand break creation is required for normal synapsis in Saccharomyces cerevisiae and the mouse, it is not necessary for synapsis in Drosophila and Caenorhabditis elegans. To investigate the timing of and requirements for double-strand break formation during Drosophila meiosis, we used an antibody that recognizes a histone modification at double-strand break sites, phosphorylation of HIS2AV (gamma-HIS2AV). Our results support the hypothesis that double-strand break formation occurs after synapsis. Interestingly, we detected a low (10-25% of wildtype) number of gamma-HIS2AV foci in c(3)G mutants, which fail to assemble synaptonemal complex, suggesting that there may be both synaptonemal complex-dependent and synaptonemal complex-independent mechanisms for generating double-strand breaks. Furthermore, mutations in Drosophila Rad54 (okr) and Rad51 (spnB) homologs cause delayed and prolonged gamma-HIS2AV staining, suggesting that double-strand break repair is delayed but not eliminated in these mutants. There may also be an interaction between the recruitment of repair proteins and phosphorylation.

Animals↗

PubMed1979

The influence of oxygen on the survival and yield of DNA double-strand breaks in irradiated yeast cells.

Survival and induction of DNA double-strand breaks were studied in cells of Saccharomyces cerevisiae irradiated under oxic or anoxic conditions with 30 MeV electrons. A linear relationship between DNA double-strand breakage and dose was found in both cases. The o.e.r.-value for colony forming ability was found to be 1.9 +/- 0.2, whereas the o.e.r.-value for DNA double-strand breakage was 3.0 +/- 0.1. These results are not inconsistent with the idea that DNA double-strand breaks are involved in killing of yeast cells. The frequency of induction of DNA double-strand breaks was found to be 0.74 x 10(-11) double-strand breaks per g/mol per Gy when cells were irradiated under oxygen and 0.24 x 10(-11) double-strand breaks per g/mol per Gy under nitrogen.

Cell Survival↗

PubMed2002

DNA double-strand break repair from head to tail.

DNA double-strand break repair is a complex process that requires multiple enzymatic and structural activities to rejoin or repair the broken DNA ends using one of several repair pathways. These enzymatic and structural activities include end detection, end processing and alignment of DNA ends. Recent structural and functional studies of the DNA double-strand break repair factors Mre11/Rad50, Ku70/80 and Xrcc4 show how these enzymes combine and assemble both enzymatic and structural activities in DNA double-strand break repair.

Adenosine Triphosphate↗

PubMed1989

Calculation of initial yields of single- and double-strand breaks in cell nuclei from electrons, protons and alpha particles.

A model of the DNA and electron and ion track structure computer codes are used to model damage in the DNA by direct action. This damage is converted into single-strand breaks using the method described by Charlton and Humm (1988) in which a minimum energy deposited in a critical volume of the DNA is correlated with the production of single-strand breaks. It is then assumed that if these single-strand breaks lie on opposite strands and are separated by less than a few base pairs they produce double-strand breaks. Absolute yields of both single- and double-strand breaks expressed in breaks/Gy-dalton are calculated and compared to measured yields. Good agreement is obtained for single-strand breaks while the calculated yields for double-strand breaks are greater than those measured.

Alpha Particles↗

PubMed1992

Directional recombination is initiated at a double strand break in human nuclear extracts.

The involvement of a double strand break in the initiation of homologous recombination was examined in human nuclear extracts. M13 duplex derivatives, containing inserts in the LacZ' region (producing white plaques), were cleaved by restriction enzymes and coincubated in the extracts with a circular plasmid containing the LacZ' region without insert, and unable to produce plaques. Repair was estimated by the ability to produce plaques after transfection into JM109 (recA1) bacteria. Recombination with the plasmid enhances the number of plaques and also the frequency of M13 producing blue plaques. Heterologous insertions in the region surrounding the break were analyzed for their effects on initiation of recombination. The extent of repair by recombination (number of plaques) was compared with the number of blue plaques among the repaired population. Initiation of recombination is inhibited when heterologous insertions are located at 7bp from the break, on the right side as well as on the left side. A low level of recombination is measurable for 27 bp of homology but the maximum efficiency of recombination occurred with homologies of 165 or 320 bp from the break to the heterologous insertion. At 320 bp, the extent of recombinational repair remained at a plateau level but the frequency of blue plaques progressively decreases. We have also analyzed the effect of different sizes of inserts. With longer inserts, a longer length of homology adjacent to the break is required for optimum recombination. However, the size of the insert does not affect the low level of recombination that occurred with a short homology (27 bp). The results indicate that the process is initiated at or near the break, requires homology on both sides of the break and is followed by an elongation from the double strand break to the distal regions of the DNA. Our data provide some support to the double-strand-break repair model established for meiotic recombination in yeast.

DNA Repair↗

PubMed2006

Age-dependent usage of double-strand-break repair pathways.

A DNA double-strand break (DSB) can be repaired by any of several alternative and competing mechanisms. The repaired sequences often differ from the original depending on which mechanism was used so that the cell's "choice" of repair mechanism can have profound genetic consequences. DSBs can accumulate with age , and human diseases that mimic some of the effects of aging, such as increased susceptibility to cancer, are associated with certain defects in DSB repair . The premeiotic germ cells of Drosophila provide a useful model for exploration of the connection between aging and DNA repair because these cells are subject to mortality and other age-related changes , and their DNA repair process is easily quantified. We used Rr3, a repair reporter system in Drosophila, to show that the relative usage of DSB repair mechanisms can change substantially as an organism ages. Homologous repair increased linearly in the male germline from 14% in young individuals to more than 60% in old ones, whereas two other pathways showed a corresponding decrease. Furthermore, the proportion of longer conversion tracts (>156 bp) also increased nearly 2-fold as the flies aged. These findings are relevant to the more general question of how DNA damage and repair are related to aging.

Age Factors↗

PubMed1993

Induction and repair of DNA double-strand breaks.

Induction and repair of DNA double-strand breaks (DSBs) was measured using a pulsed-field gel electrophoresis system. A cell line of methotrexate-resistant EMT-6 cells that contain numerous double-minutes (DMs) 3 million base pairs in size was employed. The electrophoretic mobility of these DMs depends on whether they have zero, one, or more than one DSB. With no DSBs the DMs remain as circles and are trapped in the origin of electrophoresis, but with one DSB the DMs migrate as a discrete band and can be detected easily through hybridization with a gene-specific probe. Using a clamped homogeneous electrical field apparatus, the induction of DSBs in the 1.5 to 12 Gy X-ray dose range is studied and is shown to be linear. Double-strand break repair following 7.5 Gy is studied, and is shown to be exponential. The kinetics of both induction and repair of DSBs induced in DM DNA was compared to the induction and repair of DSBs in chromosomal DNA and is shown to be similar. The kinetics of repair of DSBs following 7.5 Gy for cells embedded in agarose and cells in suspension is shown to be similar.

Cell Line↗

PubMed2000

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.

DNA↗

PubMed1999

Megabase chromatin domains involved in DNA double-strand breaks in vivo.

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named gamma-H2AX. An antibody prepared to the unique region of human gamma-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that gamma-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.

Animals↗

PubMed2003

The homologous chromosome is an effective template for the repair of mitotic DNA double-strand breaks in Drosophila.

In recombinational DNA double-strand break repair a homologous template for gene conversion may be located at several different genomic positions: on the homologous chromosome in diploid organisms, on the sister chromatid after DNA replication, or at an ectopic position. The use of the homologous chromosome in mitotic gene conversion is thought to be limited in the yeast Saccharomyces cerevisiae and mammalian cells. In contrast, by studying the repair of double-strand breaks generated by the I-SceI rare-cutting endonuclease, we find that the homologous chromosome is frequently used in Drosophila melanogaster, which we suggest is attributable to somatic pairing of homologous chromosomes in mitotic cells of Drosophila. We also find that Drosophila mitotic cells of the germ line, like yeast, employ the homologous recombinational repair pathway more often than imperfect nonhomologous end joining.

Animals↗

PubMed2004

DNA double strand break quantification in skin biopsies.

BACKGROUND AND PURPOSE: Following induction of double strand breaks the histone H2AX is rapidly phosphorylated at regions flanking the breaks resulting in nuclear gamma H2AX foci. The purpose of this study was to use this endogenous signalling system to quantify the in vivo response to radiation in normal tissue. PATIENTS AND METHODS: Skin biopsies were taken from prostate cancer patients undergoing radiotherapy with a curative intent. Biopsies were taken at locations corresponding to 5 different doses in the range below 1.1 Gy per fraction. Biopsies were taken from patients 30 min following the first fraction and then once again following the fraction given after the first weekend break in the treatment course. The DNA double strand breaks were visualised as gamma H2AX foci using immunohistochemistry. Images were acquired using a CCD-camera and a fluorescence microscope and the gamma H2AX foci were quantified using digital image analysis including the basic procedures of top-hat transformation, threshold setting and labelling. RESULTS: Repeated assessments of the biopsies showed a high reproducibility in quantifying the number of foci per DNA area of the nucleated cells in epidermis. The reproducibility was equally good for the two biopsy occasions. A linear dose response was observed for the epidermis in the dose region 0-1 Gy. CONCLUSIONS: We have established a method to measure the relative amount of DNA double strand breaks by detecting gamma H2AX foci in patients exposed to radiotherapy. The method provides a tool to study induction and repair of DNA double strand breaks and has the potential to predict individual radiosensitivity.

Biopsy↗

PubMed2003

Poly(ADP-ribose) polymerase (PARP-1) is not involved in DNA double-strand break recovery.

BACKGROUND: The cytotoxicity and the rejoining of DNA double-strand breaks induced by gamma-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose) polymerase (PARP-1) in DNA double-strand break repair. RESULTS: PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by gamma-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose) synthesis following gamma-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as gamma-rays and H2O2. CONCLUSIONS: The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to gamma-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose) synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

Animals↗

PubMed1994

Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.

Adenine Phosphoribosyltransferase↗

PubMed2001

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.

The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.

Acetyltransferases↗

PubMed1989

Imbalance of deoxyribonucleoside triphosphates, DNA double-strand breaks, and cell death caused by 2-chlorodeoxyadenosine in mouse FM3A cells.

The mechanism of the cytotoxic action of 2-chlorodeoxyadenosine in mouse FM3A cells was investigated. Imbalance of the dNTP pools occurred within 3 h of treatment with 20 microM 2-chlorodeoxyadenosine; the dATP and dGTP pools were depleted and the dTTP pool increased. 2-Chlorodeoxyadenosine added to the culture medium broke mature DNA strands, giving fragments of 100-200 kilobase pairs as found by orthogonal-field-alternation gel electrophoresis. DNA strand breaks, measured by this technique, were observed in the treated cells about 12 h after the addition. The cells also lost viability at about 12 h. Breaks in the single and double strands of DNA, as measured by alkaline and neutral filter elution, became evident 18 h after treatment with 20 microM 2-chlorodeoxyadenosine; there were as many single-strand breaks as would be caused by 130 rads of gamma-ray irradiation. Double-strand breaks were equivalent to those caused by 2180 rads of gamma-ray irradiation. Comparison of the ratio of single- and double-strand breaks caused by 2-chlorodeoxyadenosine to that following radiation suggested that 2-chlorodeoxyadenosine broke only double strands. Cycloheximide inhibited the breakage of DNA double strands and the cell death caused by this compound. Flow cytometric studies of cytostasis brought about by 2-chlorodeoxyadenosine in FM3A cells showed that cells accumulated in the earlier part of the S phase. 2-Chlorodeoxyadenosine decreased DNA synthesis more than RNA or protein synthesis. The breaks in double strands of DNA were probably important in the cell death caused by 2-chlorodeoxyadenosine. The intracellular dNTP imbalance may trigger these events.

Animals↗

PubMed1998

Correlation between cellular radiosensitivity and non-repaired double-strand breaks studied in nine mammalian cell lines.

PURPOSE: To test the relationship between cell killing and non-repaired DNA strand breaks both in repair proficient and deficient cell lines. MATERIALS AND METHODS: Five of the cell lines used are repair competent (CHO, CHO K1, rat rhabdomyosarcoma R1H, mouse balb and normal human fibroblasts), while four display a reduced repair capacity (scid, xrs1, xrs5, AT). Cell survival was determined by colony formation assay. The total number of strand breaks was measured by the alkaline unwinding technique and the numbers of double-strand breaks by constant-field gel electrophoresis. RESULTS: The nine cell lines showed a broad spectrum in radiosensitivity with SF2 values ranging from 0.018 to 0.58. The cell lines did not vary in the number of induced strand breaks, neither for all strand breaks nor for double-strand breaks alone. In contrast, there was a large variation in the number of non-repaired strand breaks measured 24 h after irradiation. Comparison of cell killing with the number of non-repaired breaks measured after a dose of 90 Gy showed no correlation for single-strand breaks (r2=0.29) but a fairly good correlation for double-strand breaks (r2=0.87). This correlation was found to hold both for repair proficient and deficient cell lines. CONCLUSIONS: The results obtained strongly suggest that the number of non-repaired double-strand breaks measured 24h after irradiation can be used as an indicator of cellular radiosensitivity.

Animals↗

PubMed1997

Genetic recombination through double-strand break repair: shift from two-progeny mode to one-progeny mode by heterologous inserts.

Double-strand break repair models of genetic recombination propose that a double-strand break is introduced into an otherwise intact DNA and that the break is then repaired by copying a homologous DNA segment. Evidence for these models has been found among lambdoid phages and during yeast meiosis. In an earlier report, we demonstrated such repair of a preformed double-strand break by the Escherichia coli RecE pathway. Here, our experiments with plasmids demonstrate that such reciprocal or conservative recombination (two parental DNAs resulting in two progeny DNAs) is frequent at a double-strand break even when there exists the alternative route of nonreciprocal or nonconservative recombination (two parental DNAs resulting in only one progeny DNA). The presence of a long heterologous DNA at the double-strand break, however, resulted in a shift from the conservative (two-progeny) mode to the nonconservative (one-progeny) mode. The product is a DNA free from the heterologous insert containing recombinant flanking sequences. The potential ability of the homology-dependent double-strand break repair reaction to detect and eliminate heterologous inserts may have contributed to the evolution of homologous recombination, meiosis and sexual reproduction.

DNA Damage↗