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Results for “Double stranded breaks”

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At least 145 records · Page 8Linked to original sources

PubMed2005

Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double-strand breaks rejoining pathway.

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by the non-homologous end-joining pathway relying on DNA-PK activity, but other mechanisms may promote end-joining. We previously described a DSB repair pathway that requires synapsis of DNA ends by poly(ADP-ribose) polymerase-1 (PARP-1) and ligation by the XRCC1/DNA ligase III complex (XL). Here, the repair of non-ligatable DNA ends by this pathway was examined in human cell extracts. The phosphorylation of the 5'-terminal end was shown to represent a limiting step for the repair process. Polynucleotide kinase (hPNK) was identified as the 5'-DNA kinase associated with the PARP-1-dependent end-joining pathway because (i) hPNK was co-recruited to DNA ends together with PARP-1 and XL, (ii) ligation of 5'-OH terminal breaks was compromised in hPNK-depleted extracts and restored upon addition of recombinant hPNK, and (iii) recombinant hPNK was necessary for end-joining of 5'-OH terminal breaks reconstituted with the PARP-1/XL complex. Also, using an assay enabling us to follow the ligation kinetics of each strand of a DSB, we established that the two strands at the junction can be processed and joined independently, so that one strand can be ligated without a ligatable nick on the other strand at the DSB site. Taken together these results reveal functional parallels between the PARP-1 and DNA-PK-dependent end-joining processes.

DNA↗

PubMed2000

Binding of inositol phosphate to DNA-PK and stimulation of double-strand break repair.

In mammalian cells, double-strand breaks in DNA can be repaired by nonhomologous end-joining (NHEJ), a process dependent upon Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV. Starting with HeLa cell-free extracts, which promote NHEJ in a reaction dependent upon all of these proteins, we have purified a novel factor that stimulates DNA end-joining in vitro. Using a combination of phosphorus NMR, mass spectroscopy, and strong anion exchange chromatography, we identify this factor as inositol hexakisphosphate (IP6). Purified IP6 is bound by DNA-PK and specifically stimulates DNA-PK-dependent end-joining in vitro. The involvement of inositol phosphate in DNA-PK-dependent NHEJ is of particular interest since the catalytic domain of DNA-PKcs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family.

Cell Fractionation↗

PubMed2002

Inhibition of DNA-double strand break repair by antimony compounds.

DNA double strand breaks (DSBs), induced by gamma-irradiation in Chinese hamster ovary cells, were used to examine whether antimony compounds affect the repair of DNA damage. The cells were first incubated with antimony trichloride or antimony potassium tartrate (both Sb(III)) for 2 h, and then irradiated with gamma-rays at a dose of 40 Gy. The DNA DSB was quantified with pulsed field gel electrophoresis immediately after irradiation (non-repair group) as well as at 30 min post-irradiation (repair group). The degree of repair inhibition was determined by the differences in the amount of DNA DSB between non-repair and repair groups. Both antimony compounds inhibited repair of DNA DSB in a dose dependent manner. In trichloride, 0.2 mM antimony significantly inhibited the rejoining of DSB, while 0.4 mM was necessary in potassium antimony tartrate. The mean lethal doses, D(0), for the treatment with antimony trichloride and antimony potassium tartrate, were approximately 0.21 and 0.12 mM, respectively. This indicates that the repair inhibition by antimony trichloride occurred in the dose range near D(0), but the antimony potassium tartrate inhibited the repair at doses where most cells lost their proliferating ability. This is the first report to indicate that antimony compounds may inhibit the repair of radiation-induced DNA DSB.

Animals↗

PubMed2001

The use of DNA double-strand break quantification in radiotherapy.

DNA double-strand breaks (DSB) are an important direct consequence of treating cells with ionizingradiation. A variety of evidence points toward DSBs being the key damage type linked to radiation-induced lethality. In particular, the link between DSB and chromosome breakage, which in turn closely correlates with cell death in some cell types, is strongly supportive of this concept. There has been much interest in the possibility of using measures of strand breaks as a pretreatment test of radiation response. This has largely been in the context of assessing inherent cellular sensitivity through damage induction or repair parameters. A number of studies have produced hopeful results, but overall there has been no parameter that can reliably predict radiosensitivity. This may be due to the inadequacies of the assays, but it is more likely to reflect the fact that the radiosensitivity of cells is dictated by a whole series of events; alterations in many of these can alter the overall response. In addition, it is now recognized that cell-signalling pathways form an essential part of the cellular response to damage, and these can be triggered by damage other than DSB. It is therefore possible that while DSBs are clearly important--and they may be the single most important lesion in some types--other damage types may be significant triggers of cell death pathways after ionizing radiation treatment.

Cell Cycle↗

PubMed2002

Checkpoints: how to flag up double-strand breaks.

How checkpoint pathways recognise double-strand breaks has long been a mystery. Recent studies have found that two distinct checkpoint protein complexes associate independently with chromatin at the sites of DNA damage. Why do two distinct mechanisms recognise strand lesions, and what does this tell us about the checkpoint pathways?

Cell Cycle Proteins↗

PubMed2001

The tight linkage between DNA replication and double-strand break repair in bacteriophage T4.

Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.

Bacteriophage T4↗

PubMed1996

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.

Base Sequence↗

PubMed1997

Loss of heterozygosity induced by a chromosomal double-strand break.

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (Rb) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the Rb locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.

Alleles↗

PubMed2001

Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair.

Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-homologous end-joining, which requires the DNA-dependent protein kinase (DNA-PK). DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku70 and Ku80 heterodimer. To define the activities of individual DNA-PK components in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells. DNA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppressed by KU70 disruption. We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G(2) phase cells, when sister chromatids are available for HR repair. To test whether direct interference by Ku70 with HR might explain the Ku70(-/-)/DNA-PKcs(-/-/-) radioresistance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells. The frequency of intrachromosomal HR induced by the I-SceI restriction enzyme was increased in the absence of Ku but not of DNA-PKcs. Significantly, abrogation of HR activity by targeting RAD54 in Ku70(-/-) or DNA-PKcs(-/-/-) cells caused extreme radiosensitivity, suggesting that the relative radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways. Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition.

Animals↗

PubMed2005

The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends.

When eukaryotic chromosomes undergo double strand breaks (DSBs), several evolutionarily conserved proteins, among which the MRX complex, are recruited to the break site, leading to checkpoint activation and DNA repair. The function of the Saccharomyces cerevisiae Sae2 protein, which is known to work together with the MRX complex in meiotic DSB processing and in specific mitotic DSB repair events, is only beginning to be elucidated. Here we provide new insights into the role of Sae2 in mitotic DSB repair. We show that repair by single strand annealing of a single DSB, which is generated by the HO endonuclease between direct repeats, is defective both in the absence of Sae2 and in the presence of the hypomorphic rad50s allele altering the Rad50 subunit of MRX. Moreover, SAE2 overexpression partially suppresses the rad50s single strand annealing repair defects, suggesting that the latter might arise from defective MRX-Sae2 interactions. Finally, SAE2 deletion slows down resection of an HO-induced DSB and impairs DSB end bridging. Thus, Sae2 participates in DSB single strand annealing repair by ensuring both resection and intrachromosomal association of the broken ends.

Alleles↗

PubMed2005

Huge differences in cellular radiosensitivity due to only very small variations in double-strand break repair capacity.

The DNA double-strand break (DSB) repair capacity of normal human fibroblasts was compared with that of cell lines with different genetic alterations. These cell lines are affected either in non-homologous end-joining (180BR), homology directed repair (C2352, C2395), base excision repair (CS1TAN, 46BR) or signalling (AT3, AT2BE, LFS2675, LFS2800, 95P558). Cellular radiosensitivity was determined by colony formation assay, DSB by constant-field gel electrophoresis and apoptosis was detected by caspase3 activity. For the mutated cell lines, the survival fraction at 2 Gy (SF2) varied between 0.013 and 0.49 in contrast to a variation of only 0.15-0.53 for normal fibroblasts. There was no variation in the number of initial DSB and only a small variation in the number of DSB remaining 24 h after irradiation. At 100 Gy, the latter number varied between 2 and 5 Gy-equivalents for normal fibroblasts and only between 3 and 7 Gy-equivalents for the mutated cell lines, corresponding to repair capacities of 95-98 and 93-97%, respectively. There were, however, two outliers (LFS2800, 180BR) where the number of remaining DSB was much higher with 22 and 30 Gy-equivalents, respectively. This elevated number resulted from a delayed repair and apoptotic cells. For all but these two cell lines, the relationship between the number of DSB remaining 24 h after irradiation and SF2 could be described by an identical correlation (r2 = 0.86, p < 0.0001). This result indicates that the relationship between DSB repair capacity and cellular radiosensitivity appears to be the same for normal and mutated cell lines, and that in both cases huge differences in cellular radiosensitivity result from only a very small variation in DSB repair capacity.

Apoptosis↗

PubMed2006

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.

Adenosine Triphosphate↗

PubMed2001

Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.

Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components. We find that the HDR frequency is enhanced in Ku70(-/-), XRCC4(-/-), and DNA-PKcs(-/-) cells, with the increase being particularly striking in Ku70(-/-) cells. Neither sister-chromatid exchange nor gene-targeting frequencies show a dependence on these NHEJ proteins. A Ku-modulated two-ended versus one-ended chromosome break model is presented to explain these results.

Animals↗

PubMed1998

Expansions and contractions in a tandem repeat induced by double-strand break repair.

Repair of a double-strand break (DSB) in yeast can induce very frequent expansions and contractions in a tandem array of 375-bp repeats. These results strongly suggest that DSB repair can be a major source of amplification of tandemly repeated sequences. Most of the DSB repair events are not associated with crossover. Rearrangements appear in 50% of these repaired recipient molecules. In contrast, the donor template nearly always remains unchanged. Among the rare crossover events, similar rearrangements are found. These results cannot readily be explained by the gap repair model of Szostak et al. (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) but can be explained by synthesis-dependent strand annealing (SDSA) models that allow for crossover. Support for SDSA models is provided by a demonstration that a single DSB repair event can use two donor templates located on two different chromosomes.

DNA Repair↗

PubMed2005

Capture of extranuclear DNA at fission yeast double-strand breaks.

Proper repair of DNA double-strand breaks (DSBs) is necessary for the maintenance of genomic integrity. Here, a new simple assay was used to study extrachromosomal DSB repair in Schizosaccharomyces pombe. Strikingly, DSB repair was associated with the capture of fission yeast mitochondrial DNA (mtDNA) at high frequency. Capture of mtDNA fragments required the Lig4p/Pku70p nonhomologous end-joining (NHEJ) machinery and its frequency was highly increased in fission yeast cells grown to stationary phase. The fission yeast Mre11 complex Rad32p/Rad50p/Nbs1p was also required for efficient capture of mtDNA at DSBs, supporting a role for the complex in promoting intermolecular ligation. Competition assays further revealed that microsatellite DNA from higher eukaryotes was preferentially captured at yeast DSBs. Finally, cotransformation experiments indicated that, in NHEJ-deficient cells, capture of extranuclear DNA at DSBs was observed if homologies--as short as 8 bp--were present between DNA substrate and DSB ends. Hence, whether driven by NHEJ, microhomology-mediated end-joining, or homologous recombination, DNA capture associated with DSB repair is a mutagenic process threatening genomic stability.

Base Sequence↗

PubMed2000

Radiation-induced heat-labile sites that convert into DNA double-strand breaks.

The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

Cells, Cultured↗

PubMed1991

Different oxygen enhancement ratios for induced and unrejoined DNA double-strand breaks in eukaryotic cells.

DNA double-strand breaks (DSBs) are 2.9 times more frequently induced in yeast cells exposed to sparsely ionizing 30-MeV electrons under oxic compared to anoxic conditions. The rejoining of DSBs induced under anoxic conditions was investigated under conditions allowing repair of potentially lethal damage and compared to the rejoining of DSBs induced in oxic cells. In contrast to the biphasic rejoining kinetics of DSBs induced in oxic cells, the rejoining kinetics of DSBs induced in anoxic cells is complicated by the formation of secondary DSBs. These arise during postirradiation incubation of cells, presumably as a consequence of repair processes acting on radiation-induced lesions other than DSBs. These secondary DSBs may at least partially explain the finding that a greater fraction of unrejoinable DSBs is present in cells irradiated under anoxic compared to oxic conditions. As a consequence, the oxygen enhancement ratio of the yield of the remaining DSBs is decreasing in the course of DSB rejoining.

Cell Hypoxia↗

PubMed2002

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells.

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of 137CS gamma-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation.

Animals↗