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PubMed2000

Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.

To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.

Adenosine Triphosphatases↗

PubMed1999

Role of yeast SIR genes and mating type in directing DNA double-strand breaks to homologous and non-homologous repair paths.

Eukaryotes have acquired many mechanisms to repair DNA double-strand breaks (DSBs) [1]. In the yeast Saccharomyces cerevisiae, this damage can be repaired either by homologous recombination, which depends on the Rad52 protein, or by non-homologous end-joining (NHEJ), which depends on the proteins yKu70 and yKu80 [2] [3]. How do cells choose which repair pathway to use? Deletions of the SIR2, SIR3 and SIR4 genes - which are involved in transcriptional silencing at telomeres and HM mating-type loci (HMLalpha and HMRa) in yeast [4] - have been reported to reduce NHEJ as severely as deletions of genes encoding Ku proteins [5]. Here, we report that the effect of deleting SIR genes is largely attributable to derepression of silent mating-type genes, although Sir proteins do play a minor role in end-joining. When DSBs were made on chromosomes in haploid cells that retain their mating type, sir Delta mutants reduced the frequency of NHEJ by twofold or threefold, although plasmid end-joining was not affected. In diploid cells, sir mutants showed a twofold reduction in the frequency of NHEJ in two assays. Mating type also regulated the efficiency of DSB-induced homologous recombination. In MATa/MATalpha diploid cells, a DSB induced by HO endonuclease was repaired 98% of the time by gene conversion with the homologous chromosome, whereas in diploid cells with an alpha mating type (matDelta/MATalpha) repair succeeded only 82% of the time. Mating-type regulation of genes specific to haploid or diploid cells plays a key role in determining which pathways are used to repair DSBs.

DNA Damage↗

PubMed1997

Clustering of meiotic double-strand breaks on yeast chromosome III.

In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-strand breaks (DSBs) that are repaired by interaction of the broken chromosome with its homologue. To identify a large number of DSB sites and gain insight into the control of DSB formation at both the local and the whole chromosomal levels, we have determined at high resolution the distribution of meiotic DSBs along the 340 kb of chromosome III. We have found 76 DSB regions, mostly located in intergenic promoter-containing intervals. The frequency of DSBs varies at least 50-fold from one region to another. The global distribution of DSB regions along chromosome III is nonrandom, defining large (39-105 kb) chromosomal domains, both hot and cold. The distribution of these localized DSBs indicates that they are likely to initiate most crossovers along chromosome III, but some discrepancies remain to be explained.

Chromosome Mapping↗

PubMed1992

2-deoxy-D-glucose inhibits rejoining of radiation-induced DNA double-strand breaks in yeast.

Effects of 2-deoxy-D-glucose (2-DG) on radiation-induced DNA double-strand breaks (dsb) have been studied under non-growth conditions in a respiratory-deficient strain of the yeast Saccharomyces cerevisiae. Velocity sedimentation in neutral sucrose gradients was used to measure DNA dsb. Addition of 2-DG to the liquid-holding medium (67 mM phosphate buffer, pH 5, 30 degrees C) at an equimolar concentration with glucose (50 mM) reduced the rate and extent of dsb rejoining. The inhibition of rejoining mediated by 2-DG is reversible for the majority--but not all--of the radiation-induced dsb.

DNA↗

PubMed1993

Effect of LET on chromosomal aberration yields. I. Do long-lived, exchange-prone double strand breaks play a role?

Dicentric chromosomal aberrations produced by ionizing radiation probably result from pairwise interaction of DNA double strand breaks (dsbs). It has been suggested that high LET radiation may preferentially produce a subclass of 'severe' dsbs that are long-lived and/or exchange-prone, and that it is the production of these severe dsbs which account for the increased biological effectiveness of high-LET radiation. We present a quantitative formalism to describe the induction of these severe dsbs, and the subsequent production of exchange-type chromosomal aberrations. Using a Markov model and microdosimetric methods, we conclude that dicentric production by such severe dsbs has properties similar to those observed at high LET. Specifically, at high doses, the yield is nearly linear with dose even if dsbs from different tracks interact. The model is applied to published data on dicentric aberrations produced by irradiation of human lymphocytes in vitro. Corrections for the effects of interphase death are estimated. From comparisons with the experiments we conclude that interaction of severe dsbs could make a significant contribution to the observed dicentric production at high LET and also perhaps for low doses (though not high doses) at low LET. Proximity explanations of high-LET effects continue to offer the main prospect for obtaining a unified picture of chromosomal aberration formation by all ionizing radiation types, but a hybrid model in which severe dsbs contribute to the high-LET aberration yield cannot be ruled out. If all or part of high-LET radiation damage is qualitatively different from low-LET radiation damage, as this severe dsb model may suggest, there could be far-reaching implications for the field of radiation protection.

Animals↗

PubMed1994

Induction of DNA damage in gamma-irradiated nuclei stripped of nuclear protein classes: differential modulation of double-strand break and DNA-protein crosslink formation.

The influence of chromatin proteins on the induction of DNA double-strand breaks (dsb) and DNA-protein crosslinks (dpc) by gamma-radiation was investigated. Low molecular weight non-histone proteins and classes of histones were extracted with increasing concentrations of NaCl, whereas nuclear matrix proteins were not extractable even by 2.0 M NaCl. The yield of dsb increased with progressive removal of proteins from chromatin. Whilst removal of low molecular weight non-histone proteins and histone H1 resulted in small increases in the production of dsb, removal of histones H2A/H2B, all histones, or all proteins led to 18.4, 46.4 and 55.5-fold increases in the yield of dsb, respectively, relative to irradiated cells. Therefore, both histones and non-histone proteins contribute to the radioprotection of DNA, core histones being the major radio-protectors. In contrast, depletion of chromatin proteins caused little or no effect on the induction of dpc until the chromatin was extracted with > or = 1.4 M NaCl. However, our studies indicated no direct, quantitative correlation between the removal of histones and the induction of dpc. The data support our previous conclusion that nuclear matrix protein rather than the majority of the histones are the predominant substrates for dpc production, although the involvement of a subset of tightly bound histones (H3 and H4) has not been excluded. This finding demonstrates that chromatin proteins can differentially modify the yield of two types of radiation-induced DNA lesions.

Animals↗

PubMed2003

Characteristics of gamma-H2AX foci at DNA double-strand breaks sites.

Phosphorylated H2AX (gamma-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to gamma-H2AX reveals that this highly amplified process generates nuclear foci. The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans. Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing. H2AX Delta/Delta mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects. gamma-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of gamma-H2AX formation.

Amino Acid Sequence↗

PubMed1996

[In vitro induction of primary DNA double-strand breaks in leukemic and normal blood cells by ultraviolet irradiation].

The yield of UV-induced DNA double-strand breaks was studied for white blood cells ("light" fraction) derived from peripheral blood, and from patients with lymphomas, chronic lymphoid leukemia (CLL), and chronic myeloid leukemia (CML). The method employed was constant-field electrophoresis of plug-embedded DNA in agarose gel. Characteristic dose-response curves were obtained for various cell populations. Lymphoid cells, both from healthy subjects and CLL patients, revealed less damage to DNA under UV-irradiation, whereas CML cells were much more affected. Possible interpretation of these results includes species-specific differences in UV-induced DNA damage, as well as sufficient DNA crosslinking, thus interfering with DNA dsbs detection in irradiated cells.

DNA Damage↗

PubMed1997

Developing a model of DNA replication to be used for Monte Carlo calculations that predict the sizes and shapes of molecules resulting from DNA double-strand breaks induced by X irradiation during DNA synthesis.

A Monte Carlo computer program was written to introduce double-strand breaks (DSBs) randomly into cellular DNA that is configured according to different models of DNA replication. Then, from a review of the literature using DNA fiber autoradiography and other studies relating to rates of replication of DNA that is organized in approximately 3-Mbp regions or bands, a particular model for DNA replication was developed. Using this model, Monte Carlo calculations were made to predict the types and sizes of molecules that would result from introducing DSBs into DNA when synchronous cells are irradiated in the middle of S phase. Then results of the Monte Carlo calculations were compared with migration profiles obtained by pulsed-field gel electrophoresis (PFGE) for molecular size distributions of linear DNA molecules. For these comparisons, CHO cells irradiated in S phase also were pulse-labeled at the time of irradiation with [3H]dThd for 15 min to compare the migration patterns of 3H-labeled replicating DNA with those of the mass of S-phase DNA, measured by imaging with a CCD camera. For the Monte Carlo calculations, we assumed from the reports in the literature that molecules containing replication bubbles with and without forks would be trapped in the PFGE plug. We also assumed that those molecules that are < or = 8 Mbp, both linear and with replication forks, would be released into the lane. However, approximately 75% of the 3H-labeled DNA that is released from the plug migrated much more slowly than linear molecules, which we attributed to the slow migration of 3H-labeled molecules having replication forks not attached to bubbles. The percentages of both mass of S-phase DNA and 3H-labeled replicating DNA released from the plug, as determined by PFGE, were compared with comparable values determined from Monte Carlo calculations. A DNA replication model that provides good agreement between the PFGE results and Monte Carlo calculations is described. Furthermore, Monte Carlo methodology is presented that can be used for comparing data obtained with PFGE with results of Monte Carlo calculations that are based on different models of DNA replication and different assumptions for the migration of various types of replicating molecules.

Animals↗

PubMed1992

Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

Complementation of the repair defect in hamster xrs mutants has been achieved by transfer of human chromosome 2 using the method of microcell-mediated chromosome transfer. The xrs mutants belong to ionizing radiation complementation group 5, are highly sensitive to ionizing radiation, and have an impaired ability to rejoin radiation-induced DNA double-strand breaks. Both phenotypes were corrected by chromosome 2, although the correction of radiation sensitivity was only partial. Complementation was achieved in two members of this complementation group, xrs6 and XR-V15B, derived independently from the CHO and V79 cell lines, respectively. The presence of human chromosome 2 in complemented clones was examined cytogenetically and by PCR analysis with primers directed at a human-specific long interspersed repetitive sequence or chromosome 2-specific genes. Complementation was observed in 25/27 hybrids, one of which contained only the q arm of chromosome 2. The two noncomplementing hybrids were missing segments of chromosome 2. The use of a back-selection system enabled the isolation of clones that had lost the human chromosome and these regained radiation sensitivity. Transfer of several other human chromosomes did not result in complementation of the repair defect in XR-V15B. These data show that the gene defective in xrs cells, XRCC5, which is involved in double-strand break rejoining, is located on human chromosome 2q.

Chromosome Banding↗

PubMed2006

Recruitment of the type B histone acetyltransferase Hat1p to chromatin is linked to DNA double-strand breaks.

Type B histone acetyltransferases are thought to catalyze the acetylation of the NH(2)-terminal tails of newly synthesized histones. Although Hat1p has been implicated in cellular processes, such as telomeric silencing and DNA damage repair, the underlying molecular mechanisms by which it functions remain elusive. In an effort to understand how Hat1p is involved in the process of DNA double-strand break (DSB) repair, we examined whether Hat1p is directly recruited to sites of DNA damage. Following induction of the endonuclease HO, which generates a single DNA DSB at the MAT locus, we found that Hat1p becomes associated with chromatin near the site of DNA damage. The nuclear Hat1p-associated histone chaperone Hif1p is also recruited to an HO-induced DSB with a similar distribution. In addition, while the acetylation of all four histone H4 NH(2)-terminal tail domain lysine residues is increased following DSB formation, only the acetylation of H4 lysine 12, the primary target of Hat1p activity, is dependent on the presence of Hat1p. Kinetic analysis of Hat1p localization indicates that it is recruited after the phosphorylation of histone H2A S129 and concomitant with the recombinational-repair factor Rad52p. Surprisingly, Hat1p is still recruited to chromatin in strains that cannot repair an HO-induced double-strand break. These results indicate that Hat1p plays a direct role in DNA damage repair and is responsible for specific changes in histone modification that occur during the course of recombinational DNA repair.

Acetylation↗

PubMed1997

Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions.

Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA-dependent protein kinase (DNA-PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA-PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double-strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA-PK defects in Ku70-/- embryonic stem cells can be counteracted by introducing epitope-tagged wild-type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site-specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA-PK, although two mutants showed that the roles of Ku70 in DNA-PK activation and IR repair are separate. Mutation of DNA-PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double-strand break repair.

Amino Acid Sequence↗

PubMed1988

Repair of DNA single-strand and double-strand breaks in the Chinese hamster xrs 5 mutant cell line as determined by DNA unwinding.

The DNA unwinding technique has been used to measure the induction and repair of DNA strand breaks by X-rays in the X-ray-sensitive (xrs 5) mutant and its parent CHO K1 line of Chinese hamster cells. Results show that frequency of induction of DNA strand breaks was the same for both cell lines. The repair of single-strand breaks was found to be slightly slower in xrs 5 over the first 20 min after X-ray exposure, but the level of repair of ssb reached after an incubation of 1h following X-ray exposure in xrs 5 was the same as in CHO K1. Our results also show that the rate of repair of DNA double-strand breaks in xrs 5 cells was clearly slower than that in CHO K1, supporting the conclusion of Kemp et al. (1984) who used the neutral elution technique, that xrs 5 is defective in the repair pathway of DNA double-strand breaks.

Animals↗

PubMed2006

BLM is an early responder to DNA double-strand breaks.

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a marked predisposition to cancer and elevated genomic instability. The defective protein in BS, BLM, is a member of the RecQ helicase family and is believed to function in various DNA transactions, including in replication, repair, and recombination. Here, we show that both endogenous and overexpressed human BLM accumulates at sites of laser light-induced DNA double-strand breaks within 10s and colocalizes with gammaH2AX and ATM. Like its RecQ helicase family member, WRN, the defective protein in Werner syndrome, dissection of the BLM protein revealed that its HRDC domain is sufficient for its recruitment to the damaged sites. In addition, we confirmed that the C-terminal region spanning amino acids 1250-1292 within the HRDC domain is necessary for BLM recruitment. To identify additional proteins required for the recruitment of BLM, we examined the recruitment of BLM in various mutants generated from chicken DT40 cells and found that the early accumulation of BLM was not dependent on the presence of ATM, RAD17, DNA-PKcs, NBS1, XRCC3, RAD52, RAD54, or WRN. Thus, HRDC domain in DNA helicases is a common early responder to DNA double-strand breaks, enabling BLM and WRN to be involved in DNA repair.

Adenosine Triphosphatases↗

PubMed2005

The RING finger ATPase Rad5p of Saccharomyces cerevisiae contributes to DNA double-strand break repair in a ubiquitin-independent manner.

Tolerance to replication-blocking DNA lesions is achieved by means of ubiquitylation of PCNA, the processivity clamp for replicative DNA polymerases, by components of the RAD6 pathway. In the yeast Saccharomyces cerevisiae the ubiquitin ligase (E3) responsible for polyubiquitylation of the clamp is the RING finger protein Rad5p. Interestingly, the RING finger, responsible for the protein's E3 activity, is embedded in a conserved DNA-dependent ATPase domain common to helicases and chromatin remodeling factors of the SWI/SNF family. Here, we demonstrate that the Rad5p ATPase domain provides the basis for a function of the protein in DNA double-strand break repair via a RAD52- and Ku-independent pathway mediated by the Mre11/Rad50/Xrs2 protein complex. This activity is distinct and separable from the contribution of the RING domain to ubiquitin conjugation to PCNA. Moreover, we show that the Rad5 protein physically associates with the single-stranded DNA regions at a processed double-strand break in vivo. Our observations suggest that Rad5p is a multifunctional protein that--by means of independent enzymatic activities inherent in its RING and ATPase domains--plays a modulating role in the coordination of repair events and replication fork progression in response to various different types of DNA lesions.

Adenosine Triphosphatases↗

PubMed2001

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts.

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5'-base overhang. Regardless of the 5'-end structure, all bleomycin-induced DSBs possess 3'-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of </=0.25 ng double-stranded DNA per band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires </=100 ng substrate DNA and >/=50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA.

Adenosine Triphosphate↗

PubMed2006

Double-strand breaks in the myotonic dystrophy type 1 and the fragile X syndrome triplet repeat sequences induce different types of mutations in DNA flanking sequences in Escherichia coli.

The putative role of double-strand breaks (DSBs) created in vitro by restriction enzyme cleavage in or near CGG*CCG or CTG*CAG repeat tracts on their genetic instabilities, both within the repeats and in their flanking sequences, was investigated in an Escherichia coli plasmid system. DSBs at TRS junctions with the vector generated a large number of mutagenic events in flanking sequences whereas DSBs within the repeats elicited no similar products. A substantial enhancement in the number of mutants was caused by transcription of the repeats and by the absence of recombination functions (recA-, recBC-). Surprisingly, DNA sequence analyses on mutant clones revealed the presence of only single deletions of 0.4-1.6 kb including the TRS and the flanking sequence from plasmids originally containing (CGG*CCG)43 but single, double and multiple deletions as well as insertions were found for plasmids originally containing (CTG*CAG)n (where n = 43 or 70). Non-B DNA structures (slipped structures with loops, cruciforms, triplexes and tetraplexes) as well as microhomologies are postulated to participate in the recombination and/or repair processes.

Colony Count, Microbial↗

PubMed2001

Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.

To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker.

Amino Acid Sequence↗