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PubMed2000

Arabidopsis DNA ligase IV is induced by gamma-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4.

Rejoining of single- and double-strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double-strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by gamma-irradiation, represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP-dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino-acid sequence to the human DNA ligase IV. Yeast two-hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV-binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C-terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by gamma-irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.

Amino Acid Sequence↗

PubMed2006

Double-strand break repair and homologous recombination in Schizosaccharomyces pombe.

The study of double-strand break repair and homologous recombination in Saccharomyces cerevisiae meiosis has provided important information about the mechanisms involved. However, it has become clear that the resulting recombination models are only partially applicable to repair in mitotic cells, where crossover formation is suppressed. In recent years our understanding of double-strand break repair and homologous recombination in Schizosaccharomyces pombe has increased significantly, and the identification of novel pathways and genes with homologues in higher eukaryotes has increased its value as a model organism for double-strand break repair. In this review we will focus on the involvement of homologous recombination and repair in different aspects of genome stability in Sz. pombe meiosis, replication and telomere maintenance. We will also discuss anti-recombination pathways (that suppress crossover formation), non-homologous end-joining, single-strand annealing and factors that influence the choice and prevalence of the different repair pathways in Sz. pombe.

DNA Breaks, Double-Stranded↗

PubMed1988

Exponential or shouldered survival curves result from repair of DNA double-strand breaks depending on postirradiation conditions.

The yeast mutant rad54-3 is temperature conditional for the rejoining of DNA double-strand breaks, but cells do proliferate at both the restrictive and permissive temperatures. Thus, after irradiation with 30 MeV electrons, survival curves can be obtained which may or may not involve double-strand break rejoining under certain experimental conditions. Because of this special property of rad54-3 cells, it was possible to demonstrate that rejoining of radiation-induced double-strand breaks under nongrowth conditions yields exponential survival curves the slopes of which decrease as a function of the rejoining time. These survival data suggest that, under nongrowth conditions, the rejoining of double-strand breaks is an unsaturated process and lacks binary misrepair. In contrast, whenever rejoining of double-strand breaks occurs under growth conditions, shouldered survival curves are observed. This is true for immediate plating as well as for delayed plating survival curves. It is proposed that it is the unsaturated rejoining of double-strand breaks under nongrowth conditions, lacking binary misrepair, which is responsible for potentially lethal damage repair.

Cell Line↗

PubMed1986

Double-strand breaks can initiate meiotic recombination in S. cerevisiae.

We investigated the effects of double-strand breaks on meiotic recombination in yeast. A double-strand break was introduced at the MATa locus by sporulation of a MAT alpha inc/MATa diploid under inducing conditions for the HO-encoded endonuclease; 14% of the resulting tetrads had undergone 4 alpha:0a conversion. Conversion at MAT was associated with co-conversion of a closely linked marker and an increased recombination frequency for flanking markers. We also studied the sporulation products of a diploid heterozygous at the HIS4 locus for an insertion of a 100 bp fragment of MATa containing the HO endonuclease cut site. Under inducing conditions, a significant number of tetrads were formed that had undergone gene conversions in favor of the HIS4+ allele. Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.

DNA↗

PubMed1979

X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution.

This work presents a neutral filter elution method for detecting DNA double strand breaks in mouse L1210 cells after X-ray. The assay will detect the number of double strand breaks induced by as little as 1000 rad of X-ray. The rate of DNA elution through the filters under neutral conditions increases with X-ray dose. Certain conditions for deproteinization, pH, and filter type are shown to increase the assay's sensitivity. Hydrogen peroxide and Bleomycin also induce apparent DNA double strand breaks, although the ratios of double to single strand breaks vary from those produced by X-ray. The introduction of double strand cuts by HpA I restriction endonuclease in DNA lysed on filters results in a rapid rate of elution under neutral conditions, implying that the method can detect double strand breaks if they exist in the DNA. The eluted DNA bands with a double stranded DNA marker in cesium chloride. This evidence suggests that the assay detects DNA double strand breaks. L1210 cells are shown to rejoin most of the DNA double strand breaks induced by 5-10 krad of X-ray with a half-time of about 40 minutes.

Animals↗

PubMed2000

Induction and repair of DNA double-strand breaks under irradiation and microgravity.

The influence of microgravity on induction and repair of double-strand breaks was studied in the yeast mutant rad54-3, which is temperature-conditional for the repair of DNA double-strand breaks. The experiment was performed on the shuttle Atlantis flight STS-84. Cell samples were kept at 0-4 degrees C until they reached orbit, where they were transferred to 22 (permissive temperature for repair) and 37 degrees C (restrictive temperature). They were exposed to graded doses of beta particles from an in-built (63)Ni source during the repair period. After 152 h in microgravity, the radiation exposure was stopped, and the samples were returned to low-temperature conditions, where they remained until final evaluation in the home laboratory. The amount of double-strand breaks remaining was estimated from the differences in survival after plating and incubation at the restrictive temperature. The results show that there is no significant difference for both the induction and the repair of double-strand breaks between microgravity and terrestrial conditions.

DNA Damage↗

PubMedDate not supplied

Caged single and double strand breaks.

Ionizing radiation and radiomimetic drugs such as bleomycin, calichieamycin, neocarzinostatin chromophore, and other synthetic agents can produce both single and double strand breaks in DNA. The ability to study the structure-activity relationships of single and double-strand break repair, lethality, and mutagenesis in vivo is complicated by the numerous types and sites of DNA cleavage products that can be induced by such agents. The ability to "cage" such breaks in DNA might help to further such studies and additionally afford a mechanism for activating and deactivating nucleic acid based drugs and probes. The major type of single strand break induced by ionizing radiation is a 3'- and 5'-phosphate terminated single nucleotide gap. Previously, a caged strand break of this type had been developed that was designed to produce the 5'-phosphate directly upon irradiation with 366 nm light, and the 3'-phosphate by a subsequent beta-elimination reaction [Ordoukhanian, P., and Taylor, J.-S. (1995) J. Am. Chem. Soc. 117, 9570]. Unfortunately, the release of the 3'-phosphate group was quite slow at pH 7. To circumvent this problem, a second caged strand break has been developed that produces the 3'-phosphate directly upon irradiation, and the 5'-phosphate by a subsequent beta-elimination reaction. When this caged strand break was used in tandem with the previous caged strand break, 5'- and 3'-phosphate terminated gaps could be directly produced by irradiation with 366 nm light. These caged single strand breaks were also incorporated in tandem into hairpin substrates to demonstrate that they could be used to cage double strand breaks. These caged single strand breaks should be generally useful for generating site-specific DNA single and double strand breaks and gaps, using wavelengths and doses of light that are nondetrimental to biological systems. Because the position of the single strand break can be varied, it should now be possible to examine the effect of the sequence context and cleavage pattern of single and double strand breaks on the lethality and mutagenicity of this important class of DNA damage.

DNA↗

PubMed1992

Influence of chromatin structure on the induction of DNA double strand breaks by ionizing radiation.

Pulsed field gel electrophoresis was used to examine the influence of chromatin structure on the induction of DNA double strand breaks by gamma-irradiation in CHO-WBL cells, nuclei, and a series of protein-depleted chromatin substrates. We developed a method to isolate intact nuclei in agarose plugs that avoids DNA shearing and nucleolytic degradation during sample preparation, and facilitates nuclear protein extraction. Agarose plug-isolated nuclei are extracted with increasing concentrations of NaCl to selectively strip off: (a) nonhistone chromosomal proteins (NHP); (b) NHP and histone H1; (c) NHP, H1, and histone H2A-H2B dimers; or (d) NHP, H1, and H2A-H2B dimers and histone H3-H4 tetramers. Following treatment with up to 40 Gy of gamma-radiation, DNA from each sample is purified and the relative induction of DNA double strand breaks is assayed by asymmetric field inversion gel electrophoresis. At a dose of 20 Gy, removal of nonhistone proteins from nuclei results in a 3-fold increase in DNA double strand breaks, compared to intact CHO cells. Additional stripping of histone H1 results in an incremental increase in double strand break induction, whereas further removal of H2A-H2B dimers yields a greater than 10-fold increase in DNA double strand breaks compared to intact CHO cells. The dose-response profile for this latter sample is similar to that observed for purified DNA. These data indicate that distinct classes of chromosomal proteins afford the DNA with different levels of protection against gamma-ray-induced DNA double strand breaks. Thus, chromatin domains that differ in tertiary structure and protein composition may also differ in their susceptibility to DNA double strand breaks induced by ionizing radiation and, perhaps, other clastogens.

Animals↗

PubMed1993

V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair.

V(D)J recombination has been examined in several X-ray-sensitive and double-strand break repair-deficient Chinese hamster cell mutants. Signal joint formation was affected in four mutants (xrs 5, XR-1, V-3, and XR-V9B cells, representing complementation groups 1 through 4, respectively) defective in DNA double-strand break rejoining. Among these four, V-3 and XR-V9B were the most severely affected. Only in V-3 was coding joint formation also affected. Ataxia telangiectasia-like hamster cell mutants (V-E5 and V-G8), which are normal for double-strand break repair but are X ray sensitive, were normal for all aspects of the V(D)J recombination reaction, indicating that X-ray sensitivity is not the common denominator but that the deficiency in double-strand break repair appears to be. The abnormality at the signal joints consisted of an elevated incidence of nucleotide loss from each of the two signal ends. Interestingly, in complementation groups 1 (xrs 5) and 2 (XR-1), signal joint formation was within the normal range under some transfection conditions. This suggests that the affected gene products in these two complementation groups are not catalytic components. Instead, they may be either secondary or stochiometric components involved in the later stages of both the V(D)J recombination reaction and double-strand break repair. The fact that such factors can affect the precision of the signal joint has mechanistic implications for V(D)J recombination.

Animals↗

PubMed1988

Cell cycle effect on the induction of DNA double-strand breaks by X rays.

Filter elution was used to compare X-ray-induced DNA single- and double-strand breaks in proliferating (P) and quiescent (Q) cells of the 66 and 67 mouse mammary tumor lines. There was no difference either between cell type or between growth states in the amount of single-strand breaks as defined by elution at pH 12.2. In contrast, Q cells appeared to sustain a much larger amount of double-strand break damage per Gray than P cells, when the damage was measured by elution at either pH 7.2 or pH 9.6. Experiments which combined centrifugal elutriation with pH 7.2 elution demonstrated that G1-P cells were similar to Q (greater than or equal to 95% G1) cells in the induction of elution-detectable double-strand breaks, while the S-phase enriched fractions sustained less damage than G1-P, Q, or asynchronous P populations. Studies in which P populations were pulse labeled with [14C]thymidine confirmed this finding. Mathematical analysis of the elution kinetics of irradiated P, Q, and S-phase cells supports a model in which the complex elution profiles observed for P cells could be explained as the sum of the one-component exponential elution profiles of G1- and S-phase subpopulations. Also, the correlation between damage measured by pH 7.2 elution and cell survival was tested by examining the dose response for stimulated 66 cells (St4), which like Q cells are greater than or equal to 95% in G1 but are more resistant to X-ray-induced cytotoxicity than are the 66 Q cells. However, the induction of double-strand breaks in St4 cells was identical to that in Q cells. Thus we conclude that there is not necessarily a correlation between the amount of elution-detectable X-ray-induced double-strand breaks and cell survival.

Animals↗

PubMed1998

Double-strand break repair mediated by DNA end-joining.

DNA double-strand breaks formed by ionizing irradiation or other stresses are repaired by homologous recombination or DNA end-joining. This review focuses on the mechanism of double-strand break repair mediated by DNA end-joining, in which many factors have recently been identified. After DNA double-strand breakage, DNA end-joining takes place between the DNA ends that have nonhomologous sequences or very short regions ofhomology. The broken DNA is repaired if the DNA end-joining occurs in the same molecule, while it causes chromosome aberrations such as deletions, insertions, translocations and inversions if it occurs between different molecules. Rad50 and its relatives, Ku-proteins, DNA ligase VI and silencing factors, are involved in DNA end-joining in yeast and mammalian cells. These findings led us to propose a model in which the formation of a heterochromatin-like complex at broken ends is an important element in DNA end-joining.

Animals↗

PubMed2006

Role of Elg1 protein in double strand break repair.

The inaccurate repair of DNA double-strand breaks (DSBs) can result in genomic instability, and additionally cell death or the development of cancer. Elg1, which forms an alternative RFC-like complex with RFC2-5, is required for the maintenance of genome stability in Saccharomyces cerevisiae, and its function has been linked to DNA replication or damage checkpoint response. Here, we show that Elg1 is involved in homologous recombination (HR)-mediated DSB repair. Mutants of elg1 were partially defective in HR induced by methylmethanesufonate (MMS) and phleomycin. Deletion of ELG1 resulted in less efficient repair of phleomycin-induced DSBs in G2/M phase-arrested cells. During HR between MAT and HML loci, Elg1 associated with both the MAT locus near the HO endonuclease-induced DSB site, and the HML homologous donor locus. The association of Elg1 with the MAT locus was not dependent on Rad52. However, Elg1 association with the HML locus depended on Rad52. Importantly, we found that two of the later steps in HR-mediated repair of an HO endonuclease-induced DSB, primer extension after strand invasion and ligation, were less efficient in elg1 mutants. Our results suggest that Elg1 is involved in DSB repair by HR.

Carrier Proteins↗

PubMed1988

Intra-chromosomal gene conversion induced by a DNA double-strand break in Saccharomyces cerevisiae.

We have stimulated mitotic and meiotic gene conversion between non-tandem direct repeats of ADE4 by a defined double-strand break imparted in vivo to one of two copies of the gene. The experimental design permitted us to distinguish unambiguously between reciprocal intra-chromosomal crossing over and non-reciprocal break-join events that could accompany the induced conversions. We observed that (1) less than 10% of the induced conversion events are accompanied by intra-chromosomal crossing over in both mitosis and meiosis; (2) non-reciprocal break-join is not stimulated by the double-strand breaks; (3) a double-strand break in meiosis is repaired off intra-chromosomal homology (if available) with approximately sevenfold preference over repair off the homologous chromosome. Our observations, analyzed in the light of previous investigations of spontaneous inter and intra-chromosomal crossing over and gene conversion, lead to the view that chromosomal configuration constrains intra-chromosomal crossing over accompanying conversion between closely spaced repeated genes during resolution of the conversion intermediate.

Chromosomes↗

PubMed1975

The repair of double-strand breaks in chromosomal DNA of yeast.

Ionizing radiation induces double-strand breaks in the nuclear DNA of the yeast Saccharomyces cerevisiae with an efficiency of approximately 0.6 X 10(-10) breaks being repaired. Based on the efficiency of break production, the sensitivity of a rad52 mutant, and the absence of radiation-induced recombination in such a mutant, it is proposed that the corresponding gene product may be involved in double-strand break repair. A model involving recombination and DNA synthesis is described for this type of repair.

Chromosomes↗

PubMed1997

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.

Adenine Phosphoribosyltransferase↗

PubMed2000

Repair of double-strand breaks by incorporation of a molecule of homologous DNA.

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double-strand breaks in the T7 genome. The efficiency of double-strand break repair was markedly increased by DNA molecules ('donor' DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending approximately 1 kb on either side of the break site. Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited. When the donor DNA molecules were labelled with 32P, repaired genomes incorporated label only near the site of the double-strand break. When repair was carried out with unlabelled donor DNA and [32P]-dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome. Repair was performed using donor DNA that had adjacent BamHI and PstI sites. When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double-strand break. These observations are most consistent with a model for double-strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.

Bacteriophage T7↗

PubMed1984

Enzymatic restriction of mammalian cell DNA using Pvu II and Bam H1: evidence for the double-strand break origin of chromosomal aberrations.

Permeabilized Chinese hamster cells were treated with the restriction enzymes Pvu II and Bam H1 which generate blunt-ended with cohesive-ended double-strand breaks in the DNA respectively. Cells were then allowed to progress to the first mitosis, where chromosomal aberrations were scored. It was found that blunt-ended double-strand breaks induced both chromosome and chromatid aberrations of exchange and deletion types, including a high frequency of tri-radials. The total aberration frequency at high enzyme concentrations was more than ten times the control background frequency. Treatment with Bam H1 on the other hand did not induce aberrations above the background rate. This may indicate that the cohesive ends generated by this enzyme may be easily repaired by the cell due to the stabilization of the hydrogen bonding at the site of the double-strand break. Measurements using the unwinding method showed that the enzymes caused strand breaks in the DNA of permeabilized cells, and an approximate X-ray dose equivalent of the restriction-enzyme-induced breaks could be calculated. This indicated that restriction-induced blunt-ended double-strand breaks are relatively inefficient in causing chromosomal aberrations. This may be because of the presence of 'clean ends' at the site of a double-strand break, which may be repaired by ligation. The method of introducing restriction enzymes into cells opens up a new model approach for the study of the conversion of double-strand breaks into chromosome aberrations.

Animals↗

PubMed2000

Meiotic double-strand breaks in Schizosaccharomyces pombe.

Meiotic DNA double-strand breaks (DSBs) are associated with recombination hot spots in the yeast Saccharomyces cerevisiae and are believed to initiate the process of recombination. Until now, meiosis-induced breaks have not been shown to occur regularly in other organisms. Here we show, by pulsed-field gel electrophoresis of DNA, that meiotic DSBs occur transiently in all three chromosomes of the fission yeast Schizosaccharomyces pombe. In a repair defective mutant, carrying a mutation in the RecA homolog gene rhp51, meiotic DSBs accumulate. In contrast to expectation from the genetic map of S. pombe, however, many chromosomal DNA molecules remain unbroken during meiosis.

Chromosomes, Fungal↗