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Results for “Double stranded breaks”

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PubMed1994

Effects of heavy ions on inactivation and DNA double strand breaks in Deinococcus radiodurans R1.

Inactivation and double strand break (dsb) induction after heavy ion irradiation were studied in stationary phase cells of the highly radiation resistant bacterium Deinococcus radiodurans R1. There is evidence that the radiation sensitivity of this bacterium is nearly independent on energy in the range of up to 15 MeV/u for lighter ions (Ar). The responses to dsb induction for charged particles show direct relationship between increasing radiation dose and residual intact DNA.

Argon↗

PubMed2004

Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break.

BACKGROUND: In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form gamma-H2AX) and accumulate repair proteins at or near DSBs. To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between gamma-H2AX formation and repair protein recruitment remains to be defined. RESULTS: We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae. Break induction provoked rapid gamma-H2AX formation and equally rapid recruitment of the Mre11 repair protein. gamma-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most gamma-H2AX formation was dependent on Tel1 and Mre11. gamma-H2AX formed in a large (ca. 50 kb) region surrounding the DSB. Remarkably, very little gamma-H2AX could be detected in chromatin within 1-2 kb of the break. In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break. CONCLUSIONS: Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.

Chromatin↗

PubMed2004

DNA double-strand break repair by homologous recombination.

DNA double-strand breaks (DSB) are presumed to be the most deleterious DNA lesions as they disrupt both DNA strands. Homologous recombination (HR), single-strand annealing, and non-homologous end-joining are considered to be the pathways for repairing DSB. In this review, we focus on DSB repair by HR. The proteins involved in this process as well as the interactions among them are summarized and characterized. The main emphasis is on eukaryotic cells, particularly the budding yeast Saccharomyces cerevisiae and mammals. Only the RAD52 epistasis group proteins are included.

DNA Damage↗

PubMed2003

Distance from the chromosome end determines the efficiency of double strand break repair in subtelomeres of haploid yeast.

Double strand break (DSB) repair plays an important role in chromosome evolution. We have investigated the fate of DSBs as a function of their location along the yeast chromosome XI, in a system where no conventional homologous recombination can occur. We report that the relative frequency of non-homologous endjoining (NHEJ), which is the exclusive mode of DSB repair in the internal chromosomal portion, decreases gradually towards the telomere, keeping the absolute frequency nearly constant, and that other repair mechanisms, which generally involve the loss of the distal chromosomal fragment, appear in subtelomeric regions. Distance of the DSB from chromosome ends plays a critical role in the global frequency of these repair mechanisms. Direct telomere additions are rare, and other events such as break-induced replication, plasmid incorporation, and gene conversion, involve acquisition of heterologous sequences. Therefore, in subtelomeric regions, cell survival to DSBs is higher and alternative modes of repair allow new genomic combinations to be generated. Furthermore, subtelomeric rearrangements depend on the recombination process, which, unexpectedly, also promotes the joining of heterologous sequences. Finally, we report that the Rad52 protein increases the efficiency of NHEJ.

Base Sequence↗

PubMed2003

PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint inactivation after a double-strand break.

Saccharomyces cells suffering a DNA double-strand break (DSB) ultimately escape checkpoint-mediated G2/M arrest either by recovery once the lesion is repaired or by adaptation if the lesion proves irreparable. Cells lacking the PP2C-like phosphatases Ptc2 and Ptc3 are unable to adapt to a HO-induced DSB and are also defective in recovering from a repairable DSB. In contrast, overexpression of PTC2 rescues adaptation-defective yku80Delta and cdc5-ad mutants. These effects are not explained by alterations either in the processing of DSB ends or in DSB repair. In vivo and in vitro evidence suggests that phosphorylated forms of Ptc2 and Ptc3 specifically bind to the Rad53 FHA1 domain and inactivate Rad53-dependent pathways during adaptation and recovery by dephosphorylating Rad53.

Cell Cycle Proteins↗

PubMed2003

Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast.

Repair of DNA double-strand breaks (DSBs) by homologous recombination requires members of the RAD52 epistasis group. Here we use chromatin immunoprecipitation (ChIP) to examine the temporal order of recruitment of Rad51p, Rad52p, Rad54p, Rad55p, and RPA to a single, induced DSB in yeast. Our results suggest a sequential, interdependent assembly of Rad proteins adjacent to the DSB initiated by binding of Rad51p. ChIP time courses from various mutant strains and additional biochemical studies suggest that Rad52p, Rad55p, and Rad54p each help promote the formation and/or stabilization of the Rad51p nucleoprotein filament. We also find that all four Rad proteins associate with homologous donor sequences during strand invasion. These studies provide a near comprehensive view of the molecular events required for the in vivo assembly of a functional Rad51p presynaptic filament.

Cells, Cultured↗

PubMed1995

Differential dependence on chromatin structure for copper and iron ion induction of DNA double-strand breaks.

The induction of DNA DSB (double-strand breaks) in isolated nuclear chromatin by Cu(II) or Fe(II)-EDTA in the presence of H2O2 and ascorbate has been compared to DSB induction by gamma-radiation. V79 nuclei embedded in agarose plugs were treated with each agent on ice, and the resultant DNA fragments were analyzed by pulsed-field gel electrophoresis. In the absence of low molecular weight radical scavengers, both irradiation and treatment with iron ion induced random DSB, as judged by the size distribution of DNA fragments, and the yield of DSB in each case was enhanced by either the expansion of chromatin (approximately 5-fold) or the removal of histones (21-25-fold) before treatment. In contrast, treatment with Cu(II) produced small DNA fragments of uniform size (approximately 100-200 kbp), independent of the yield of DSB. In addition, neither the DNA fragment size nor the yield of DSB produced by Cu(II) was affected by the prior removal of histones from chromatin. Deproteinized DNA was degraded randomly by Cu(II) but at a slower rate than observed for chromatin. In the presence of ascorbate, H2O2 was found to be essential for DSB induction by Fe(II)-EDTA but not by Cu(II), possibly because H2O2 can be produced from ascorbate and Cu(II) in the presence of oxygen. Despite the above differences between the production of DSB by the two metal ions, DSB induction in native chromatin by either metal ion was blocked by 0.1 M EDTA or 0.25 M thiourea but was resistant to the hydroxyl radical scavengers 0.25 M DMSO and 0.25 M mannitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1997

Initiation of V(D)J recombination in vivo: role of recombination signal sequences in formation of single and paired double-strand breaks.

In V(D)J recombination, double-strand breaks (DSBs) are introduced at recombination signal sequences (RSSs) which consist of three distinct elements: a heptamer, a 12 or 23 nucleotide spacer and a nonamer. Efficient DSB formation requires a 12/23 RSS pair and occurs at both RSS in a temporally coupled fashion (coupled cleavage). It remains unknown which RSS elements are important for coupled cleavage. Furthermore, it has not been established whether some RSS components are critical only for cleavage in cis, with others mainly promoting cleavage in trans at the partner RSS. We investigated these questions by analyzing the effects of RSS mutations on the formation of DSBs in vivo. The abundance of DSBs in cis (at the mutant RSS) and in trans (at the consensus RSS) was determined using an established ligation-mediated PCR assay. We also developed a Southern blotting approach that allows the first direct measurement of dual and single RSS cleavage in vivo. Our results demonstrate that the heptamer, spacer and nonamer elements are all required for coupled cleavage in vivo. These studies also provide evidence for cleavage events involving a single RSS both in mutant substrates and in substrates containing a consensus 12/23 RSS pair.

DNA↗

PubMed2001

Capture of DNA sequences at double-strand breaks in mammalian chromosomes.

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.

Animals↗

PubMed2002

Focused genetic recombination of bacteriophage t4 initiated by double-strand breaks.

A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segCDelta strain of bacteriophage T4 was developed. The ets1, a 66-bp DNA fragment of phage T2L containing the cleavage site for the T4 SegC site-specific endonuclease, was inserted into the proximal part of the T4 rIIB gene. Under segC(+) conditions, the ets1 behaves as a recombination hotspot. Crosses of the ets1 against rII markers located to the left and to the right of ets1 gave similar results, thus demonstrating the equal and symmetrical initiation of recombination by either part of the broken chromosome. Frequency/distance relationships were studied in a series of two- and three-factor crosses with other rIIB and rIIA mutants (all segC(+)) separated from ets1 by 12-2100 bp. The observed relationships were readily interpretable in terms of the modified splice/patch coupling model. The advantages of this localized or focused recombination over that distributed along the chromosome, as a model for studying the recombination-replication pathway in T4 in vivo, are discussed.

Bacteriophage T4↗

PubMed2003

Ku-dependent and Ku-independent end-joining pathways lead to chromosomal rearrangements during double-strand break repair in Saccharomyces cerevisiae.

Chromosomal double-strand breaks (DSBs) can be repaired by either homology-dependent or homology-independent pathways. Nonhomologous repair mechanisms have been relatively less well studied, despite their potential importance in generating chromosomal rearrangements. We have developed a Saccharomyces cerevisiae-based assay to identify and characterize homology-independent chromosomal rearrangements associated with repair of a unique DSB generated within an engineered URA3 gene. Approximately 1% of successfully repaired cells have accompanying chromosomal rearrangements consisting of large insertions, deletions, aberrant gene conversions, or other more complex changes. We have analyzed rearrangements in isogenic wild-type, rad52, yku80, and rad52 yku80 strains, to determine the types of events that occur in the presence or absence of these key repair proteins. Deletions were found in all strain backgrounds, but insertions were dependent upon the presence of Yku80p. A rare RAD52- and YKU80-independent form of deletion was present in all strains. These events were characterized by long one-sided deletions (up to 13 kb) and extensive imperfect overlapping sequences (7-22 bp) at the junctions. Our results demonstrate that the frequency and types of repair events depend on the specific genetic context. This approach can be applied to a number of problems associated with chromosome stability.

Antigens, Nuclear↗

PubMed1996

Effects of terminal nonhomology and homeology on double-strand-break-induced gene conversion tract directionality.

Double-strand breaks (DSBs) greatly enhance gene conversion in the yeast Saccharomyces cerevisiae. In prior plasmid x chromosome crosses, conversion tracts were often short ( < 53 bp) and usually extended in only one direction from a DSB in an HO recognition sequence inserted into ura3. To allow fine-structure analysis of short and unidirectional tracts, phenotypically silent markers were introduced at 3- and 6-bp intervals flanking the HO site. These markers, which created a 70-bp homeologous region (71% homology), greatly increased the proportion of bidirectional tracts. Among products with short or unidirectional tracts, 85% were highly directional, converting markers on only one side (the nearest marker being 6 bp from the HO site). A DSB in an HO site insertion creates terminal nonhomologies. The high degree of directionality is a likely consequence of the precise cleavage at homology/nonhomology borders in hybrid DNA by Rad1/10 endonuclease. In contrast, terminal homeology alone yielded mostly unidirectional tracts. Thus, nonhomology flanked by homeology yields primarily bidirectional tracts, but terminal homeology or nonhomology alone yields primarily unidirectional tracts. These results are inconsistent with uni- and bidirectional tracts arising from one- and two-ended invasion mechanisms, respectively, as reduced homology would be expected to favor one-ended events. Tract spectra with terminal homeology alone with similar in RAD1 and rad1 cells, indicating that the high proportion of bidirectional tracts seen with homeology flanking nonhomology is not a consequence of Rad1/10 cleavage at homology/homeology boundaries. Instead, tract directionality appears to reflect the influence of the degree of broken-end homology on mismatch repair.

Base Sequence↗

PubMed2006

The effect of gap length on double-strand break repair in Drosophila.

When a double-strand break has a gap between the broken ends, the missing information can be restored through synthesis from a homologous template. Here we address the question of how long such a gap can be before this process fails. We measured the frequency of homologous repair in the Drosophila germ line following the creation of gaps of specific sizes ranging from 3.8 to 210 kb. We found that gaps of >11 kb.

Animals↗

PubMed2006

Mechanisms of eukaryotic DNA double strand break repair.

For all cells, a DNA double strand break (DSB) is a dangerous lesion that can have profound consequences for the genome. If a DSB is encountered during mitosis, chromosomal separation may be adversely affected. Alternatively, during S phase a DSB may cause replication fork stalling or collapse. Improperly repaired DSBs can result in chromosomal rearrangements, senescence or activation of apoptotic pathways. Cells have developed sophisticated recombination pathways to metabolize and repair DSBs quickly as well as the capacity to differentiate physiologically occurring breaks from life threatening lesions. The two major pathways of recombination repair are known as non-homologous end-joining (NHEJ) and homologous recombination (HR). In this review, we will discuss the detection, response, and repair of DSBs in eukaryotes.

Antigens, Nuclear↗

PubMed2005

ATP-dependent chromatin remodeling and DNA double-strand break repair.

The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic stability. Two pathways for the repair of DBSs, nonhomologous end-joining (NHEJ) and homologous recombination (HR), have evolved in eukaryotes. These pathways, like processes such as transcription and replication, act on DNA that is embedded in nucleosomes. Recent studies have shown that DNA repair, like transcription, is facilitated both by histone tail modification and by ATP-dependent chromatin remodeling. This review emphasizes recent reports that demonstrate a function for the ATP-dependent chromatin remodeling complexes INO80 and RSC in NHEJ and HR. We also discuss the possible role of SWR1- and TIP60-mediated nucleosomal histone exchange in DNA repair.

Adenosine Triphosphate↗

PubMed1994

Computer simulation of pulsed field gel runs allows the quantitation of radiation-induced double-strand breaks in yeast.

A procedure for the quantification of double-strand breaks in yeast is presented that utilizes pulsed field gel electrophoresis (PFGE) and a comparison of the observed DNA mass distribution in the gel lanes with calculated distributions. Calculation of profiles is performed as follows. If double-strand breaks are produced by sparsely ionizing radiation, one can assume that they are distributed randomly in the genome, and the resulting DNA mass distribution in molecular length can be predicted by means of a random breakage model. The input data for the computation of molecular length profiles are the breakage frequency per unit length, alpha, as adjustable parameter, and the molecular lengths of the intact chromosomes. The obtained DNA mass distributions in molecular length must then be transformed into distributions of DNA mass in migration distance. This requires a calibration of molecular length vs. migration distance that is specific for the gel lane in question. The computed profiles are then folded with a Lorentz distribution with adjusted spread parameter gamma to account for band broadening. The DNA profiles are calculated for different breakage frequencies alpha and for different values of gamma, and the parameters resulting in the best fit of the calculated to the observed profile are determined.

Computer Simulation↗

PubMed1990

Effects of 3'-deoxyadenosine (cordycepin) on the repair of X-ray-induced DNA single- and double-strand breaks in Chinese hamster V79 cells.

The ability of cordycepin to inhibit the repair of DNA strand breaks was examined with X-irradiated Chinese hamster V79 cells in log-phase culture. A filter elution technique revealed that 70 microM cordycepin did not inhibit the repair of single-strand breaks but inhibited the repair of double-strand breaks. These findings confirmed the fact that the increase in the lethality of cordycepin in X-irradiated cultured mammalian cells was attributable to unrepaired DNA double-strand breaks.

Animals↗

PubMed1981

Repair of DNA double-strand breaks in Escherichia coli cells requires synthesis of proteins that can be induced by UV light.

The repair of DNA double-strand breaks in Escherichia coli cells irradiated with gamma rays occurs only after new proteins are synthesized in response to damage introduced in the genome DNA. One protein whose synthesis is thus induced is the recA protein, and previous work has shown that recA- cells do not repair double-strand breaks. However, inducing recA protein by treating cells with nalidixic acid does not induce repair of double-strand breaks, so this repair requires more than the presence of the recA protein. When repair of double-strand breaks is blocked, the genome DNA is degraded by an endonuclease-like action. Evidence is presented to show that the inducible inhibition of DNA degradation after x-irradiation [Pollard, E. C. & Randall, E. P. (1973) Radiat. Res. 55, 265] is probably caused by the inducible repair of DNA double-strand breaks.

Bacterial Proteins↗