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PubMed1993

Genetic analysis of double-strand break repair in Escherichia coli.

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.

Bacterial Proteins↗

PubMed1989

Structure of bleomycin-induced DNA double-strand breaks: predominance of blunt ends and single-base 5' extensions.

In order to examine the structure of bleomycin-induced DNA double-strand breaks, defined-sequence DNA was labeled in each strand at a single restriction site and treated with bleomycin. Various double-stranded fragments resulting from bleomycin-induced double-strand breaks were isolated, denatured, and run on sequencing gels to determine the sites of cleavage in each strand. For virtually every double-strand break, the cleavage site in one strand was a pyrimidine in a G-Py sequence, reflecting a specificity similar to that of bleomycin-induced single-strand cleavage. However, the cleavage site in the complementary strand was seldom a G-Py sequence, and was usually a site where single-strand cleavage was infrequent. When the sequence at the double-strand break was G-Py-Py', the break at Py was usually accompanied by a break at the base directly opposite Py, resulting in blunt ends. When the sequence was G-Py-Pu, the break at Py was usually accompanied by a break at the base opposite Pu, resulting in single-base 5' extensions. Double-strand breaks with 3' extensions, such as would result from cleavage of two C residues in a self-complementary G-C sequence, were conspicuously absent. These data provide further evidence that bleomycin-induced double-strand breaks do not result from coincidence of independent site-specific single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiotics, Antineoplastic↗

PubMed1984

S1 nuclease-sensitive sites in yeast DNA: an assay for radiation-induced base damage.

DNA has been isolated from gamma-irradiated yeast cells and then treated with the enzyme S1 nuclease. This enzyme cleaves DNA specifically at sites where localized denaturation has occurred and can therefore be used as a lesion probe to identify regions in the DNA where base-pairing has been disrupted. By analysing the number of single strand breaks, double strand breaks and alkali-labile sites in the DNA before and after treatment with S1 nuclease, it has been possible to calculate the number of S1 nuclease-sensitive sites induced as a result of exposure to ionizing radiation. These sites were found to occur with a frequency about twice that of the double strand break and are thought to result from a primary ionization event in the DNA.

Cobalt Radioisotopes↗

PubMed1986

Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells.

The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed.

Centrifugation, Density Gradient↗

PubMed1992

Structural effect of donor DNA on the initiation of recombination for double strand break repair in human nuclear extracts.

The effect of the structure of donor DNA molecules on the initiation of recombination for double strand break repair in human nuclear extracts, was investigated here. A unique double strand break was introduced into M13 duplex derivatives by digestion with restriction enzymes. After coincubation of the cleaved DNA in human nuclear extracts, with a plasmid containing M13 sequences spanning the break, double strand break repair was estimated by the plating efficiency in JM109 (RecA1) bacteria. We first confirm that a short heterologous insert (8bp) close to the break on the recipient cleaved M13 DNA inhibits recombination with circular as well as with linear donor molecules. The results indicate that, with these substrates, recombination is initiated at the level of the break, requires uninterrupted homology on both sides of the break, and is associated with a decreasing gradient of gene conversion. When the heterologous insertion is located on the plasmid donor DNA, similar results are obtained with a circular donor DNA. In contrast, with a linear donor molecule, bearing the insert, homology requirements, in the region of the break in M13 DNA, are abolished. This last result suggests that recombination could be initiated at the extremities of the linear donor DNA.

Bacteriophage M13↗

PubMed2004

Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks.

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.

Ataxia Telangiectasia Mutated Proteins↗

PubMed1986

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes.

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.

Adult↗

PubMed1999

Quantitative assessment of the contribution of clustered damage to DNA double-strand breaks induced by 60Co gamma rays and fission neutrons.

The induction of DNA strand breaks by fission neutrons was studied in aqueous plasmid (pBR322) DNA under aerobic conditions for a wide range of hydroxyl radical (*OH) scavenger concentrations and was compared to the induction of strand breaks by 6OCo gamma rays. Strand breaks were measured using agarose gel electrophoresis coupled with sensitive 32P-based phosphor imaging. Yields are reported for DNA single-strand breaks (SSBs) and double-strand breaks formed linearly with dose (alphaDSBs). The fraction of alphaDSBs that were dependent on the multiply damaged site (MDS) or clustered damage mechanism was also calculated using a model. G values for SSBs and alphaDSBs declined with increasing *OH scavenging capacity. However, with increasing *OH scavenging capacities, the decrease in yields of strand breaks for fission neutrons was not as pronounced as for gamma rays. The percentage of alphaDSBs for gamma rays was dependent on *OH scavenging capacity, appearing negligible at low scavenging capacities but increasing at higher scavenging capacities. In contrast, fission neutrons induced high percentages of alphaDSBs that were approximately independent of *OH scavenging capacity. The levels of alphaDSBs formed by the MDS mechanism after exposure to fission neutrons are consistent with the expected distinctive features of high-LET energy deposition events and track structure. The results also confirm observations made by others that even for low-LET radiation, the MDS mechanism contributes significantly to DNA damage at cell-like scavenging conditions.

Cobalt Radioisotopes↗

PubMed1988

Induction and rejoining of gamma-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells and in two radiosensitive clones.

The induction and rejoining of gamma-ray-induced DNA strand breaks were measured in a Chinese hamster ovary cell line, AA8, and in two radiosensitive clones (EM9 and NM2) derived from it. The kinetics of recovery from sublethal damage (SLD) and potentially lethal damage (PLD) has previously been characterized in each of these lines [vanAnkeren et al., Radiat. Res., 115, 223-237 (1988)]. No significant differences were observed among the cell lines in the yields of either DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) as assayed by filter elution. Data for SSB rejoining in AA8 and NM2 cells irradiated with 7.5 Gy were fit by a biexponential process (t1/2 values of approximately 4 and 80 min). In comparison, SSB rejoining in EM9 cells was initially slower (t1/2 = 10 min) and a higher level of SSBs was unrejoined 6 h after irradiation. DSB rejoining in AA8 cells assayed at pH 9.6 was also biphasic (t1/2 values of 15 and 93 min), although when assayed at pH 7.0, most (approximately 80%) of the damage was rejoined at a constant rate (t1/2 = 45 min) during the first 2 h. EM9 cells exhibited a slower initial rate of DSB rejoining when assayed at pH 9.6 but showed no difference compared with AA8 cells in DSB rejoining when assayed at pH 7.0. These results indicate that radiosensitive EM9 cells, whose kinetics of recovery from SLD and PLD was the same as that of AA8 cells, have a defect in the fast phase of SSB rejoining but no measurable defect in DSB rejoining. Conversely, NM2 cells, which displayed a reduced shoulder width on their survival curve and decreased recovery from SLD, had no demonstrable defects in the rate or extent of rejoining of DSBs or SSBs. When compared with the SLD and PLD data reported previously, these results suggest that there is no direct correlation between either of these recovery processes and the rejoining of SSBs or DSBs as assayed here.

Animals↗

PubMed1983

The oxygen enhancement ratio for single- and double-strand breaks induced by tritium incorporated in DNA of cultured human T1 cells. Impact of the transmutation effect.

The effect of oxygen, expressed as the oxygen enhancement ratio (OER), on the number of single-strand breaks (SSB) and double-strand breaks (DSB) induced in DNA by the radioactive decay of tritium was measured in human T1 cells whose DNA had been labeled with tritium at carbon atom number 6 of thymidine. Decays were accumulated in vivo under aerobic conditions at 0-1 degrees C and at -196 degrees C and in a nitrogen atmosphere at 0-1 degrees C. The number of SSB and DSB produced was analyzed by sucrose gradient centrifugation. For each tritium decay there were 0.25 DSB in cells exposed to air at 0-1 degrees C and 0.07 in cells kept under nitrogen, indicating an OER of 3.6, a value expected for such low-LET radiation. However, for each tritium decay there were 1.25 SSB in cells exposed to air at 0-1 degrees C and 0.76 in cells kept under nitrogen indicating an OER of only 1.7. The corresponding values for 60Co gamma radiation, expressed as SSB per 100 eV absorbed energy, were 4.5 and 1.0, giving an OER of 4.5. The low OER value found for SSB induced by tritium decay can be explained if 31% of the total SSB produced in air result from transmutation by a mechanism which does not produce DSB and is unaffected by oxygen.

Cell Line↗

PubMed2003

Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes.

DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci. Here we show that gammaH2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting gammaH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced gammaH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This gammaH2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in gammaH2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form gammaH2AX. However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved gammaH2AX response for double-strand breaks induced by replication fork collision. gammaH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.

Animals↗

PubMed2006

Biochemical mechanisms of chromosomal translocations resulting from DNA double-strand breaks.

Exposure of mammalian cells to agents that induce DNA double-strand breaks typically results in both reciprocal and nonreciprocal chromosome translocations. Over the past decade, breakpoint junctions of a significant number of translocations and other genomic rearrangements, both in clinical tumors and in experimental models, have been analyzed at the DNA sequence level. Based on these data, reasonable inferences regarding the biochemical mechanisms involved in translocations can be drawn. In a few cases, breakpoints have been shown to correlate with sites of double-strand cleavage by agents to which the cells or patients have been exposed, including exogenous rare-cutting endonucleases, radiomimetic compounds, and topoisomerase inhibitors. These results confirm that translocations primarily reflect misjoining of the exchanged ends of two or more double-strand breaks. Many junctions show significant loss of DNA sequence at the breakpoints, suggesting exonucleolytic degradation of DNA ends prior to joining. The size and frequency of these deletions varies widely, both between experimental systems, and among individual events in a single system. Homologous recombination between repetitive DNA sequences does not appear to be a major pathway for translocations associated with double-strand breaks. Rather, the general features of the junction sequences, particularly the high frequency small terminal deletions, the apparent splicing of DNA ends at microhomologies, and gap-filling on aligned double-strand break ends, are consistent with the known biochemical properties of the classical nonhomologous end joining pathway involving DNA-dependent protein kinase, XRCC4 and DNA ligase IV. Nevertheless, cells with deficiencies in this pathway still exhibit translocations, with grossly similar junction sequences, suggesting an alternative but less conservative end joining pathway. Although evidence for participation of specific DNA end processing enzymes in formation of translocations is largely circumstantial, likely candidates include DNA polymerases lambda and mu, Artemis nuclease, polynucleotide kinase/phosphatase, tyrosyl-DNA phosphodiesterase, DNase III, Werner syndrome protein, and the Mre11/Rad50/NBS1 complex.

Animals↗

PubMed2026

MRE11 suppresses germline mutagenesis at meiotic double-strand breaks in mice.

SPO11 forms hundreds of double-strand breaks (DSBs) to initiate meiotic recombination that is normally error-free. However, SPO11 activity can be mutagenic when one chromatid incurs closely spaced DSBs (double cuts), especially when DSBs are dysregulated by loss of the ATM kinase. De novo indels and structural variants can arise via end joining at double cuts within a single hotspot (microdeletions) or at adjacent hotspots separated by at least 30 kb, as we now show, sometimes accompanied by ectopic insertions of double-cut fragments. Here, we investigate how meiotic DSB end processing influences end joining. In MRE11-deficient mouse spermatocytes, which do not resect their DSBs, deletions at double cuts occur readily, with end-joining breakpoint profiles closely matching SPO11 DSB profiles. Microdeletions suggest that two DSBs can be as close as ∼21 bp. The tyrosyl-DNA phosphodiesterase TDP2 contributes to both deletion formation and ectopic insertion of double-cut fragments, presumably by removing SPO11 from DNA ends prior to joining. Finally, observations suggest a cooperative role for MRE11 and ATM in locally regulating DSB distributions. Our findings provide insight into the mechanism of de novo mutation origin, emphasizing the role of meiotic DSBs in shaping genome evolution.

Animals↗

PubMed1994

One-sided invasion events in homologous recombination at double-strand breaks.

Two classes of homologous recombination mechanism for repair of double-strand breaks (DSBs) have been described in eukaryotes so far. One is conservative and has been explained by the double-strand break repair model (Szostak et al., 1983), whereas the other one is non-conservative and has been explained by the single-strand annealing model (Lin et al., 1984). Here, we will review data supporting the existence of another homologous recombination mechanism for double-strand break repair. We will present the one-sided invasion model that we have proposed to explain this mechanism and discuss its potential implication in various homologous recombination events.

Animals↗

PubMed2003

Deletion, rearrangement, and gene conversion; genetic consequences of chromosomal double-strand breaks in human cells.

Chromosomal double-strand breaks (DSBs) in mammalian cells are usually repaired through either of two pathways: end-joining (EJ) or homologous recombination (HR). To clarify the relative contribution of each pathway and the ensuing genetic changes, we developed a system to trace the fate of DSBs that occur in an endogenous single-copy human gene. Lymphoblastoid cell lines TSCE5 and TSCER2 are heterozygous (+/-) or compound heterozygous (-/-), respectively, for the thymidine kinase gene (TK), and we introduced an I-SceI endonuclease site into the gene. EJ for a DSB at the I-SceI site results in TK-deficient mutants in TSCE5 cells, while HR between the alleles produces TK-proficient revertants in TSCER2 cells. We found that almost all DSBs were repaired by EJ and that HR rarely contributes to the repair in this system. EJ contributed to the repair of DSBs 270 times more frequently than HR. Molecular analysis of the TK gene showed that EJ mainly causes small deletions limited to the TK gene. Seventy percent of the small deletion mutants analyzed showed 100- to 4,000-bp deletions with a 0- to 6-bp homology at the joint. Another 30%, however, were accompanied by complicated DNA rearrangements, presumably the result of sister-chromatid fusion. HR, on the other hand, always resulted in non-crossing-over gene conversion without any loss of genetic information. Thus, although HR is important to the maintenance of genomic stability in DNA containing DSBs, almost all chromosomal DSBs in human cells are repaired by EJ.

Chromosomes↗

PubMed1995

The quality of DNA double-strand breaks: a Monte Carlo simulation of the end-structure of strand breaks produced by protons and alpha particles.

The quality of DNA damage induced by protons and alpha-particles of various linear energy transfer (LET) was studied. The aim was to single out specific lesions in the DNA molecule that might lead to biological endpoints such as inactivation. A DNA model coupled with a track structure code (MOCA-15) were used to simulate the lesions induced on the two helixes. Four categories of DNA breaks were considered: single-strand breaks (ssb), blunt-ended double-strand breaks (dsb, with no or few overlapping bases), sticky-ended double-strand breaks (with cohesive free ends of many bases), and deletions (complex lesions which involve at least two dsb within a small number of base pairs). Calculations were carried out assuming various sets of parameters characterizing the production of these different DNA breaks. No large variations in the yields of ssb and blunt- or sticky-ended dsb were found in the LET range between 10 and 200 keV/mu m. On the other hand, the yield of deletions increases up to about 100 keV/mu m and seems to reach a plateau at higher LET values. In the LET interval from 30 to 60 keV/mu m, protons proved to be more efficient than alpha-particles in inducing deletions. The induction of these complex lesions is thus dependent not simply on LET but also on the characteristics of the track structure. Comparison with RBE values for cell killing shows that this special class of dsb might play an important role in radiation-induced cell inactivation.

Alpha Particles↗

PubMed1991

Significance and measurement of DNA double strand breaks in mammalian cells.

Techniques for measuring DNA double strand breaks in mammalian cells are being used increasingly by researchers studying both physiological processes, such as recombination, replication, and apoptosis, as well as pathological processes, such as clastogenesis induced by ionizing radiation, chemotherapeutic drugs, and chemical toxicants. In this review we evaluate commonly used assays for measuring DNA double strand breaks, focusing on neutral filter elution and pulsed field gel electrophoresis, and explore the advantages and limitations of applying these techniques to problems of current interest in carcinogenesis and genetic toxicology.

Animals↗

PubMed1994

Radiation-induced mitotic gene conversion frequency in yeast is modulated by the conditions allowing DNA double-strand break repair.

Repair of DNA double-strand breaks (DSB) involves recombinational processes which may lead to gene conversion (intragenic recombination). Using the diploid yeast mutant rad54-3 heteroallelic for his1 (his1-7/his1-1) and temperature conditional for DSB rejoining, radiation induced gene conversion was investigated as dependent on DSB repair under different postirradiation conditions. Gene conversion is negligible under conditions preventing DSB repair (36 degrees C). In contrast, gene conversion is observed when cells are incubated at the permissive temperature (23 degrees C) both under growth and nongrowth conditions. However, there is a much higher yield of convertants for cells incubated under growth as opposed to nongrowth conditions. These results can most plausibly be explained by the cell cycle regulated enhancement of the expression of genes such as PMS and POL3 known to be involved in gene conversion processes and/or the enhanced recombination in transcriptionally active genes. 'Nutrient stress' inducible responses and/or cell cycle specific recombination pathways leading to gene conversion events preferentially in S-phase cells seem to be less likely.

Cell Survival↗