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PubMed2004

Molecular mechanism of the recruitment of NBS1/hMRE11/hRAD50 complex to DNA double-strand breaks: NBS1 binds to gamma-H2AX through FHA/BRCT domain.

DNA double-strand breaks represent the most potentially serious damage to a genome, and hence, many repair proteins are recruited to DNA damage sites by as yet poorly characterized sensor mechanisms. We clarified that NBS1 physically interacts with gamma-H2AX to form nuclear foci at DNA damage sites. The fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT) of NBS1 are essential for this physical interaction and focus formation of NBS1 in response to DNA damage. The inhibition of this interaction by introduction of anti-gamma-H2AX antibody into cells abolishes NBS1 foci formation in response to DNA damage. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for re-localization of the NBS1/hMRE11/hRAD50 complex to the vicinity of DNA damage. Moreover, the foci formation of DNA repair-related proteins containing BRCT domain, such as BRCA1, requires the interaction with gamma-H2AX in response to DNA damage. These findings indicate that the physical interaction between gamma-H2AX and DNA repair-related proteins is indispensable for the recruitment of these proteins. Further, it was recently reported that the NBS1/hMRE11/hRAD50 complex has a crucial role for both the recruitment of ATM to DNA damage sites and the subsequent activation of ATM. Therefore, both gamma-H2AX and the NBS1/hMRE11/hRAD50 complex might function for the initial recognition of DNA damage.

Acid Anhydride Hydrolases↗

PubMed1998

Indirect mechanisms contribute to biological effects produced by decay of DNA-incorporated iodine-125 in mammalian cells in vitro: double-strand breaks.

We have examined whether nuclear DNA can be protected from double-strand breaks (DSBs) induced by decay of the Auger-electron-emitting radionuclide 125I. Decays were accumulated at 0.3 degrees C in Chinese hamster V79 cells suspended in isotonic buffer containing 0.1 M EDTA in the presence or absence of 10% dimethyl sulfoxide (DMSO). DSBs were measured by the neutral elution method (pH 9.6) and quantified as strand scission factors. DMSO was shown to protect DNA from DSBs caused by the decay of DNA-incorporated 125I. The dose modification factor (DMF) for this radionuclide decreases as a function of 125I decays (389 to 4,100 decays, DMF = 2.5 to 1.3). Extrapolation of the curve for the DMF indicates that at approximately 15,000 decays/cell, a DMF of 1 would be obtained. Experiments using large numbers of 125I decays confirmed these extrapolations. For induction of DSBs by 137Cs gamma rays, the DMF also decreases with dose (50 to 290 Gy, DMF = 2.7 to 1.5). However, extrapolation of the curve for the DMF indicates that protection does not cease at higher doses. The data show that, at the same level of damage, DMSO can protect against gamma-ray-induced DSBs 1.35-fold more efficiently than against DSBs caused by the decay of DNA-incorporated 125I. It appears that when 125I is incorporated into DNA, chromatin structure fosters some DSB formation by an indirect mechanism(s) and that more than one DSB is generated per decaying atom.

Animals↗

PubMed1994

Requirement for double-strand breaks but not for specific DNA sequences in herpes simplex virus type 1 genome isomerization events.

Herpes simplex virus type 1 (HSV-1) genome isomerization occurs as a result of DNA replication-mediated homologous recombination between several sets of inverted repeat sequences present in the viral DNA. The frequency with which this recombination occurs has been demonstrated to be dependent upon DNA homology length rather than specific sequences. However, the smallest of the viral inverted repeats, the alpha sequence, has been shown to function as a recombinational hot spot, leading to speculation that this sequence may represent a specific element through which genome isomerization is mediated. To investigate this apparent paradox, a quantitative transient recombination assay system was developed and used to examine the recombinogenic properties of a panel of alpha sequence mutants. This analysis revealed that the presence of both the pac1 and pac2 elements was both necessary and sufficient for the induction of high-frequency recombination events by the alpha sequence. However, it was the double-strand break promoted by pac1 and pac2 during cleavage and packaging at the alpha sequence, and not the DNA sequences of the elements themselves, which appeared to be critical for recombination. This was illustrated (i) by the inability of the same pac1 and pac2 sequences to mediate inversion events in cells infected with an HSV-1 mutant which was competent for DNA replication-dependent recombination but defective for the cleavage and packaging process and (ii) by the ability of double-strand breaks generated in non-HSV-1 DNA by an in vivo-expressed restriction endonuclease to significantly stimulate the initiation of recombination events in virus-infected cells. Thus, the alpha sequence appears to act as a hot spot for homologous recombination simply because it happens to coincide with the site of the double-strand break which is generated during the cleavage and packaging process, not because it contains discrete sequences which are required for this activity. However, it was found that this enhanced recombinogenicity disappeared when the element was flanked by regions of extensive sequence homology, particularly that of the large inverted repeats which flank the alpha sequence at its natural site in the HSV-1 genome. These findings are consistent with a model for HSV-1 genome isomerization in which recombination is initiated primarily by multiple random double-strand breaks which arise during DNA replication across the inverted repeats of the genome, rather than by a single specific break which occurs at the alpha sequence during the cleavage and packaging process.

Animals↗

PubMed2003

Schizosaccharomyces pombe Rdh54 (TID1) acts with Rhp54 (RAD54) to repair meiotic double-strand breaks.

We report the characterization of rdh54+, the second fission yeast Schizosaccharomyces pombe Rad54 homolog. rdh54+ shares sequence and functional homology to budding yeast RDH54/TID1. Rdh54p is present during meiosis with appropriate timing for a meiotic recombination factor. It interacts with Rhp51 and the meiotic Rhp51 homolog Dmc1 in yeast two-hybrid assays. Deletion of rdh54+ has no effect on DNA damage repair during the haploid vegetative cell cycle. In meiosis, however, rdh54Delta shows decreased spore viability and homologous recombination with a concomitant increase in sister chromatid exchange. The rdh54Delta single mutant repairs meiotic breaks with similar timing to wild type, suggesting redundancy of meiotic recombination factors. Consistent with this, the rdh54Delta rhp54Delta double mutant fails to repair meiotic double strand breaks. Live cell analysis shows that rdh54Delta rhp54Delta asci do not arrest, but undergo both meiotic divisions with near normal timing, suggesting that failure to repair double strand breaks in S. pombe meiosis does not result in checkpoint arrest.

Amino Acid Sequence↗

PubMed2005

Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo.

Antibacterial quinolones inhibit type II DNA topoisomerases by stabilizing covalent topoisomerase-DNA cleavage complexes, which are apparently transformed into double-stranded breaks by cellular processes such as replication. We used plasmid pBR322 and two-dimensional agarose gel electrophoresis to examine the collision of replication forks with quinolone-induced gyrase-DNA cleavage complexes in Escherichia coli. Restriction endonuclease-digested DNA exhibited a bubble arc with discrete spots, indicating that replication forks had been stalled. The most prominent spot depended upon the strong gyrase binding site of pBR322, providing direct evidence that quinolone-induced cleavage complexes block bacterial replication forks in vivo. We differentiated between stalled forks that do or do not contain bound cleavage complex by extracting DNA under different conditions. Resealing conditions allow gyrase to efficiently reseal the transient breaks within cleavage complexes, while cleavage conditions cause the latent breaks to be revealed. These experiments showed that some stalled forks did not contain a cleavage complex, implying that gyrase had dissociated in vivo and yet the fork had not restarted at the time of DNA isolation. Additionally, some branched plasmid DNA isolated under resealing conditions nonetheless contained broken DNA ends. We discuss a model for the creation of double-stranded breaks by an indirect mechanism after quinolone treatment.

Anti-Infective Agents↗

PubMed1994

Meiosis-induced double-strand break sites determined by yeast chromatin structure.

Double-strand DNA breaks (DSBs) occur at recombination hotspots during Saccharomyces cerevisiae meiosis and are thought to initiate exchange at these loci. Analysis of DSB sites in three regions of the yeast genome indicated that breaks occur at or near many potential transcription promoters and that DSBs initiate most, if not all, meiotic recombination. DSB sites displayed deoxyribonuclease I hypersensitivity in chromatin from mitotic and meiotic cells, and changes in chromatin structure produced parallel changes in the occurrence of DSBs. Thus, features of chromatin structure that are established before meiosis play a role in determining where meiotic recombination events initiate.

Chromatin↗

PubMed1989

dNTP imbalance and DNA double strand breaks in mouse FM3A cells and the mechanism of cell death.

The mechanism of intracellular deoxyribonucleoside-triphosphate (dNTP) imbalance death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to 5-fluorodeoxyuridine, deoxyadenosine, or 2-chlorodeoxyadenosine, dNTP pool imbalance resulted. The imbalance was followed by DNA double-strand breaks and subsequent cell death. The DNA double strand breaks were directly examined by means of orthogonal-field-alternation gel electrophoresis (OFAGE). Fragmented DNA band appeared to be approximately 100-200 kbp in size. The bases of 5'-termini in the DNA were cytosine and thymine. The imbalance induced endonuclease has been isolated by DEAE-agarose column chromatography.

2-Chloroadenosine↗

PubMed1995

Recombination of Ty elements in yeast can be induced by a double-strand break.

The Ty retrotransposons are the main family of dispersed repeated sequences in the yeast Saccharomyces cerevisiae. These elements are flanked by a pair of long terminal direct repeats (LTRs). Previous experiments have shown that Ty elements recombine at low frequencies, despite the fact that they are present in 30 copies per genome. This frequency is not highly increased by treatments that cause DNA damage, such as UV irradiation. In this study, we show that it is possible to increase the recombination level of a genetically marked Ty by creating a double-strand break in it. This break is repaired by two competing mechanisms: one of them leaves a single LTR in place of the Ty, and the other is a gene conversion event in which the marked Ty is replaced by an ectopically located one. In a strain in which the marked Ty has only one LTR, the double-strand break is repaired by conversion. We have also measured the efficiency of repair and monitored the progression of the cells through the cell-cycle. We found that in the presence of a double-strand break in the marked Ty, a proportion of the cells is unable to resume growth.

Base Sequence↗

PubMed1977

Repair of DNA double-strand breaks requires two homologous DNA duplexes.

DNA repair and cell survival in haploid and its diploid derivative strains of Saccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in the G1 stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.

DNA Repair↗

PubMed1993

Fluorometric determination of DNA in agarose gels: usefulness for measurement of double-strand breaks in nonlabeled cells by pulsed-field gel electrophoresis.

Quantitative measurement of DNA in agarose gels, particularly as needed for measurement of double-strand breaks induced by agents such as radiation, usually involves the use of radioactively labeled DNA. Thus its usefulness is usually limited to growing cells which incorporate radiolabeled thymidine into DNA. To circumvent this problem, we have developed a fluorometric technique for quantitative estimation of DNA in the presence of large amounts of agarose. Gel slices are solubilized with concentrated sodium perchlorate and DNA is selectively precipitated with cadmium chloride. The amount of DNA can then be estimated with 3,5-diaminobenzoic acid. Determination of DNA is linear in the range 10 ng to 1 microgram or more. We describe the application of this technique to the measurement of 60Co gamma-ray-induced double-strand breaks by pulsed-field gel electrophoresis. Our results are essentially identical to those obtained using radiolabeled DNA.

Aminobenzoates↗

PubMed2006

Activation-induced cytidine deaminase acts on double-strand breaks in vitro.

Activation-induced cytidine deaminase (AID) is likely responsible for DNA cytidine deamination, although it may also act as an RNA deaminase. It functions on single-stranded DNA, the non-template strand in double-stranded DNA during transcription, or both strands in supercoiled DNA. To ask whether AID is able to deaminate cytidine at DNA breaks, plasmids, containing a SnaBI site (TAC downward arrowGTA) that forms blunt ends after digestion with SnaBI, were generated. If AID deaminates cytidine at the upstream blunt end, the ATG start codon in either of two drug resistance genes will be regenerated after ligation and replication in UDG-null E. coli cells. This study shows that AID targets cytidine at the break. The extent of deamination activity beyond the break is correlated with the base composition in the break region. If the break region is A, T-rich, C > T transitions are extensive. However, when the break region is not A, T-rich, mutations are mainly restricted to the break, similar to findings in vivo. The results indicate that AID has activity on double strand breaks (DSBs). Based on previous and current findings, a somatic hypermutation (SHM) model is proposed, in which collision between the transcription apparatus and the replication fork generates DSBs. After AID acts on break ends, the error-prone DNA repair machinery fixes and creates mutations.

Ampicillin Resistance↗

PubMed1992

Evidence that DNA double-strand breaks initiate the phenotype of delayed reproductive death in Chinese hamster ovary cells.

A persistently reduced cloning efficiency occurs in many of the cloned progeny of Chinese hamster ovary (CHO) cells surviving X irradiation, a stable phenotype we have previously termed delayed reproductive death (Int. J. Radiat. Biol. 60, 483-496, 1991). We now report that this phenotype is also induced by the alkylating agent ethyl methanesulfonate (EMS), but not by irradiation with ultraviolet light. The restriction endonuclease HinfI, which binds at G [symbol: see text] ANTC DNA sequences and generates cohesive-end double-strand breaks, was also efficient in inducing delayed reproductive death. On the other hand, an X-ray-sensitive CHO mutant, xrs-5, which is defective in the repair of DNA double-strand breaks, did not show this phenotype following X irradiation. These results suggest that DNA double-strand breaks, as well as the endogenous repair processes for these breaks, are associated with the induction of the delayed reproductive death phenotype in CHO cells. The possible mechanism for the induction of delayed reproductive death by EMS is discussed.

Animals↗

PubMed1994

Lack of a correlation between radiosensitivity and DNA double-strand break induction or rejoining in six human tumor cell lines.

The neutral filter elution method and the neutral comet assay have been used to analyze radiation-induced DNA damage and repair in 6 human tumor cell lines: HT-144 melanoma; DU-145 prostate carcinoma; U-87 glioma; WiDr and HT-29 colon adenocarcinomas; and SiHa cervical carcinoma. In spite of large differences in intrinsic radiosensitivity measured using a clonogenic assay, double-strand break induction, rejoining rate, and amount of residual DNA damage 4 h after irradiation were similar among these cell lines when measured using the neutral comet assay. Differences in initial numbers of double-strand breaks were observed using the neutral filter elution method; however, there was no correlation with radiosensitivity, nor did the rejoining rate or amount of residual DNA damage measured using filter elution correlate with radiation sensitivity. We conclude that neither double-strand break assay is able to reliably rank cells according to clonogenic survival following irradiation.

Cell Survival↗

PubMed1983

Repair-induced DNA double strand breaks after ultraviolet-light and either aphidocolin or 1-beta-D-arabinofuranosylcytosine/hydroxyurea.

We recently showed that the DNA repair processes occurring after u.v. light induce double strand breaks (DSBs) in the DNA of normal human fibroblast (IMR-90) but not of xeroderma pigmentosum group A cells. We call these lesions 'repair-induced double strand breaks' (RDSBs), since they only occur in excision repair competent cells. In this work, we asked whether these RDSBs occur in IMR-90 cells at low u.v. doses and whether the RDSBs are themselves repairable. We reasoned that holding open the excision-repair induced gaps by inhibiting nucleotide polymerization after u.v. light with hydroxyurea/ara C or aphidocolin should allow us to answer these questions. The results show that as little as 2.5 J.m-2 of u.v. light induces RDSBs during repair incubation when repair inhibitors are present. This suggests that 'hot spots' of high lesion frequency occur and the overlapping excision in these areas will produce RDSBs. Without repair inhibitors, the frequency of such RDSBs will be much lower; but even one unrepaired RDSB is likely to have profound biological effects. By removing aphidocolin from the medium after incubation of RDSBs, we were able to test the repairability of these lesions. The results show that RDSBs are only partially repairable with between 15 and 40% of the breaks unrepaired at 24 h. Because the lesions are partially repairable they should not always cause toxicity and may be involved in processes such as mutation, transformation, and chromosome or chromatid type aberrations of the sort associated with human tumors.

Aphidicolin↗

PubMed1996

The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair.

The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication. Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants. The spa-47 suppressor mutation maps within or very near dnaC. These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E. coli involve extensive DNA replication.

Bacterial Proteins↗

PubMed2006

Phosphorylation of H2AX histones in response to double-strand breaks and induction of premature chromatin condensation in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia faba, and Allium porrum.

Histone H2A variant H2AX is rapidly phosphorylated on the induction of DNA double-strand breaks by ionizing radiation and hydroxyurea-mediated replication arrest, resulting in the formation of gamma-H2AX foci along megabase chromatin domains nearby the sites of incurred DNA damage. In an attempt to establish a relationship between species-specific nuclear architecture and H2AX phosphorylation in S/G(2) phase-arrested root meristem cells, immunocytochemical comparisons using an antibody raised against human gamma-H2AX were made among three plants differing with respect to DNA contents: Allium porrum, representing a reticulate type of DNA package, Vicia faba, having semireticulate cell nuclei, and Raphanus sativus, characterised by a chromocentric type of chromatin. Another approach was aimed at determining possible correlations between the extent of hydroxyurea-induced phosphorylation of H2AX histones and the quantities of root meristem cells induced by caffeine to enter aberrant mitotic division (premature chromosome condensation). It was concluded that the higher-order structure of chromatin may contribute to the accessibility of molecular factors engaged in the recognition and repair of genetic lesions. Consequently, in contrast to A. porrum and V. faba, a diffuse chromatin in chromocentric cell nuclei of R. sativus may become more vulnerable both to generate DNA double-strand breaks and to recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus, more resistant to factors which allow the cells to enter premature chromosome condensation spontaneously. On the other hand, however, caffeine-mediated overriding of the S-M checkpoint control system resulted in the typical appearance of premature chromosome condensation, irrespective of the genomic content of DNA.

Antineoplastic Agents↗

PubMed1986

Inviability of dam recA and dam recB cells of Escherichia coli is correlated with their inability to repair DNA double-strand breaks produced by mismatch repair.

The molecular basis for the inviability of dam-3 recA200(Ts) and dam-3 recB270(Ts) cells was studied. The dam-3 recA200(Ts) cells were inviable in yeast extract-nutrient broth or in minimal medium at 42 degrees C. Although the dam-3 recB270(Ts) cells were inviable in yeast extract-nutrient broth at 42 degrees C, they were viable at 42 degrees C in minimal medium, in which the high salt content suppresses the mutant phenotype caused by the recB270(Ts) mutation at 42 degrees C. Under the growth conditions rendering dam rec cells inviable, the cells accumulated double-strand breaks in their DNA. Introduction of a mutL or mutS mutation restored the viability of dam-3 recB270(Ts) cells grown in yeast extract-nutrient broth at 42 degrees C and eliminated the formation of DNA double-strand breaks in these cells. We conclude that the inability to repair DNA double-strand breaks produced by the mismatch repair process accounts for the inviability of the dam recA and dam recB cells.

Culture Media↗

PubMed1993

Recombination initiated by double-strand breaks.

The HO endonuclease was used to introduce a site-specific double-strand break (DSB) in an interval designed to monitor mitotic recombination. The interval included the trp1 and his3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Mitotic recombination was monitored in a diploid carrying heteroalleles of trp1 and his3. The normal recognition sites for the HO endonuclease were mutated at the MAT alleles and a synthetic recognition site for HO endonuclease was placed between trp1 and his3 on one of the chromosomes. HO-induced cleavage resulted in efficient recombination in this interval. Most of the data can be explained by double-strand gap repair in which the cut chromosome acts as the recipient. However, analysis of some of the recombinants indicates that regions of heteroduplex were generated flanking the site of the cut, and that some recombinants were the result of the cut chromosome acting as the genetic donor.

Base Sequence↗