Double-strand Break Repair Kinetics Research Articles

Mouse HORMAD1 Is a Meiosis I Checkpoint Protein That Modulates DNA Double-Strand Break Repair During Female Meiosis1

Oocytes in embryonic ovaries enter meiosis I and arrest in the diplonema stage. Perturbations in meiosis I, such as abnormal double-strand break (DSB) formation and repair, adversely affect oocyte survival. We previously discovered that HORMAD1 is a critical component of the synaptonemal complex but not essential for oocyte survival. No significant differences were observed in the number of primordial, primary, secondary, and developing follicles between wild-type and Hormad1(−/−)newborn, 8-day, and 80-day ovaries. Meiosis I progression in Hormad1(−/−) embryonic ovaries was normal through the zygotene stage and in oocytes arrested in diplonema; however, we did not visualize oocytes with completely synapsed chromosomes. We investigated effects of HORMAD1 deficiency on the kinetics of DNA DSB formation and repair in the mouse ovary. We irradiated Embryonic Day 16.5 wild-type and Hormad1(−/−) ovaries and monitored DSB repair using gammaH2AX, RAD51, and DMC1 immunofluorescence. Our results showed a significant drop in unrepaired DSBs in the irradiated Hormad1(−/−) zygotene oocytes as compared to the wild-type oocytes. Moreover, Hormad1 deficiency rescued Dmc1(−/−) oocytes. These results indicate that Hormad1 deficiency promotes DMC1-independent DSB repairs, which in turn helps asynaptic Hormad1(−/−) oocytes resist perinatal loss.

Evaluation of the Gamma-H2AX Assay for Radiation Biodosimetry in a Swine Model

There is a paucity of large animal models to study both the extent and the health risk of ionizing radiation exposure in humans. One promising candidate for such a model is the minipig. Here, we evaluate the minipig for its potential in γ-H2AX-based biodosimetry after exposure to ionizing radiation using both Cs137 and Co60 sources. γ-H2AX foci were enumerated in blood lymphocytes and normal fibroblasts of human and porcine origin after ex vivo γ-ray irradiation. DNA double-strand break repair kinetics in minipig blood lymphocytes and fibroblasts, based on the γ-H2AX assay, were similar to those observed in their human counterparts. To substantiate the similarity observed between the human and minipig we show that minipig fibroblast radiosensitivity was similar to that observed with human fibroblasts. Finally, a strong γ-H2AX induction was observed in blood lymphocytes following minipig total body irradiation. Significant responses were detected 3 days after 1.8 Gy and 1 week after 3.8 and 5 Gy with residual γ-H2AX foci proportional to the initial radiation doses. These findings show that the Gottingen minipig provides a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans.

Biochemical DSB-repair model for mammalian cells in G1 and early S phases of the cell cycle

The paper presents a model of double strand breaks (DSB) repair in G1 and early S phases of the cell cycle. The model is based on a plethora of published information on biochemical modification of DSB induced by ionizing radiation. So far, three main DSB repair pathways have been identified, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). During G1 and early S phases of the cell cycle, NHEJ and MMEJ repair pathways are activated dependent on the type of double strand breaks. Simple DSB are a substrate for NHEJ, while complex DSB and DSB in heterochromatin require further end processing. Repair of all DSB start with NHEJ presynaptic processes, and depending on the type of DSB pursue simple ligation, further end processing prior to ligation, or resection. Using law of mass action the model is translated into a mathematical formalism. The solution of the formalism provides the step by step and overall repair kinetics. The overall repair kinetics are compared with the published experimental measurements. Our calculations are in agreement with the experimental results and show that the complex types of DSBs are repaired with slow repair kinetics. The G1 and early S phase model could be employed to predict the kinetics of DSB repair for damage induced by high LET radiation.

The Non-homologous End-Joining (NHEJ) Mathematical Model for the Repair of Double-Strand Breaks: II. Application to Damage Induced by Ultrasoft X Rays and Low-Energy Electrons

We investigated the kinetics of simple and complex types of double-strand breaks (DSB) using our newly proposed mechanistic mathematical model for NHEJ DSB repair. For this purpose the simulated initial spectrum of DNA DSB, induced in an atomistic canonical model of B-DNA by low-energy single electron tracks, 100 eV to 4.55 keV, and the electrons generated by ultrasoft X rays (CK, AlK and TiK), were subjected to NHEJ repair processes. The activity elapsed time of sequentially independent steps of repair performed by proteins involved in NHEJ repair process were calculated for separate DSB. The repair kinetics of DSBs were computed and compared with published data on repair kinetics obtained by pulsed-field gel electrophoresis method. The comparison shows good agreement for V79-4 cells irradiated with ultrasoft X rays. The average times for the repair of simple and complex DSB confirm that double-strand break complexity is a potential explanation for the slow component of DSB repair observed in V79-4 cells irradiated by ultrasoft X rays.

The PARP3- and ATM-dependent phosphorylation of APLF facilitates DNA double-strand break repair

APLF is a forkhead associated-containing protein with poly(ADP-ribose)-binding zinc finger (PBZ) domains, which undergoes ionizing radiation (IR)-induced and Ataxia-Telangiectasia Mutated (ATM)-dependent phosphorylation at serine-116 (Ser116). Here, we demonstrate that the phosphorylation of APLF at Ser116 in human U2OS cells by ATM is dependent on poly(ADP-ribose) polymerase 3 (PARP3) levels and the APLF PBZ domains. The interaction of APLF at sites of DNA damage was diminished by the single substitution of APLF Ser116 to alanine, and the cellular depletion or chemical inhibition of ATM or PARP3 also altered the level of accumulation of APLF at sites of laser-induced DNA damage and impaired the accumulation of Ser116-phosphorylated APLF at IR-induced γH2AX foci in human cells. The data further suggest that ATM and PARP3 participate in a common signalling pathway to facilitate APLF-Ser116 phosphorylation, which, in turn, appears to be required for efficient DNA double-strand break repair kinetics and cell survival following IR. Collectively, these findings provide a more detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage and suggest that Ser116-APLF phosphorylation facilitates APLF-dependent double-strand break repair.

An Inverse Switch in DNA Base Excision and Strand Break Repair Contributes to Melphalan Resistance in Multiple Myeloma Cells

Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.

Abstract 1451: DAB2IP, a novel tumor suppressor protein modulates prostate cancer cell death in response to the combined treatment of ionizing radiation and a DNA-PKcs inhibitor NU7441

Abstract Introduction: Prostate cancer (PCa) is the most common malignancy (25%) and the second leading cause of cancer-related death (10%) in U.S. men. Radiation treatment, which is noninvasive and well tolerated, is one of the front line therapeutic interventions in the management of localized PCa. However, significant proportions of high-risk patients fail therapy and develop metastatic disease. DAB2IP, which is a novel member of the Ras GTPase-activating protein family and a regulator of PI3K/Akt activity, is often downregulated and associated with aggressive prostate cancer (PCa). The purpose of this study is to investigate the radio-sensitizing property of a novel DNA-PKcs inhibitor, NU7441, in DAB2IP- deficient human PCa cells. Methods: We have developed DAB2IP deficient and proficient PCa cell lines (C4-2, PC3) for this study. We performed clonogenic surviving fraction analysis, DNA double strand break repair kinetics, cell cycle analysis, TUNEL assays, immunofluorescence, immunoblot, apoptosis and autophagy assays to investigate the mechanism of radiosensitization of NU7441 in these PCa cells. Results: Clonogenic surviving fraction analysis indicates a strong radiosensitizing ability of NU7441 in PCa cells deficient in DAB2IP. This is primarily due to robust G2-M block and persistent DNA double strand break in response to the combined treatment of radiation and NU7441. We clearly demonstrate that the presence of DAB2IP modulates prostate cancer cell death when treated with radiation and NU7441. Apoptotic cell death was prominent in PCa cells proficient in DAB2IP whereas, robust autophagic cell death was noticed in DAB2IP deficient PCa cells. Conclusion: Our study clearly shows that NU7441, a novel DNA-PKcs inhibitor, is a potent radiosensitizer in aggressive PCa cells. Furthermore, our study indicates that DAB2IP may act as an important factor in PCa cell death after combined treatment Nu7441 and radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1451. doi:1538-7445.AM2012-1451

The influence of DNA inhibitor synthesis on the induction and repair of double-strand DNA breaks in human lymphocytes under action of radiation with a different linear energy transfer

The influence that inhibitors of repair and replicative DNA synthesis, 1-β-D-arabinofuranosyl-cytosine and hydroxyurea, have on the formation and repair kinetics of double-strand breaks (DSBs) in peripheral human blood lymphocytes under the influence of radiation with a different linear energy transfer (LET) (gamma quanta and accelerated heavy ions) is studied. It is demonstrated that lithium and boron ions with LETs of 20 and 40 keV/μm, respectively, possess higher biological effectiveness with respect to the DNA DSB induction criterion. The value of the relative biological effectiveness of accelerated lithium and boron ions is 1.5 ± 0.1 and 1.6 ± 0.1, respectively. It is found that, upon cell irradiation by gamma quanta in the absence of inhibitors, efficient DNA DSB repair is observed during incubation. Under the conditions of cell incubation and in the presence of inhibitors, some growth in the number of DNA DSBs, rather than a reduction, is observed after 5-h incubation. In the case of the action of accelerated boron ions (as well as gamma quanta), under normal conditions, the efficient repair of induced DNA lesions takes place. Unlike the action of gamma quanta, in the case of cell incubation in the presence of radiomodifiers, the number of induced DNA DSBs falls. These results may testify to the fact that the repair of double-strand DNS breaks takes place under the action of ionizing radiation with a different LET on mammalian cells in the presence of DNA synthesis inhibitors Ara-C and HU. It is concluded that, for cells subject to gamma irradiation, no DNA DSB repair is observed due to the large contribution of single-strand incision DNA breaks formed in the postradiation period in the course of excision nucleotide repair.

Photosensitization by iodinated DNA minor groove binding ligands: Evaluation of DNA double-strand break induction and repair

Iodinated DNA minor groove binding bibenzimidazoles represent a unique class of UVA photosensitizer and their extreme photopotency has been previously characterized. Earlier studies have included a comparison of three isomers, referred to as ortho-, meta- and para-iodoHoechst, which differ only in the location of the iodine substituent in the phenyl ring of the bibenzimidazole. DNA breakage and clonogenic survival studies in human erythroleukemic K562 cells have highlighted the higher photo-efficiency of the ortho-isomer (subsequently designated UV A Sens) compared to the meta- and para-isomers. In this study, the aim was to compare the induction and repair of DNA double-strand breaks induced by the three isomers in K562 cells. Further, we examined the effects of the prototypical broad-spectrum histone deacetylase inhibitor, Trichostatin A, on ortho-iodoHoechst/UVA-induced double-strand breaks in K562 cells. Using γH2AX as a molecular marker of the DNA lesions, our findings indicate a disparity in the induction and particularly, in the repair kinetics of double-strand breaks for the three isomers. The accumulation of γH2AX foci induced by the meta- and para-isomers returned to background levels within 24 and 48 h, respectively; the number of γH2AX foci induced by ortho-iodoHoechst remained elevated even after incubation for 96 h post-irradiation. These findings provide further evidence that the extreme photopotency of ortho-iodoHoechst is due to not only to the high quantum yield of dehalogenation, but also to the severity of the DNA lesions which are not readily repaired. Finally, our findings which indicate that Trichostatin A has a remarkable potentiating effect on ortho-iodoHoechst/UVA-induced DNA lesions are encouraging, particularly in the context of cutaneous T-cell lymphoma, for which a histone deacetylase inhibitor is already approved for therapy. This finding prompts further evaluation of the potential of combination therapies.

Modeling DNA Double-Strand Break Repair Kinetics as an Epiregulated Cell-Community-Wide (Epicellcom) Response to Radiation Stress

The multicellular signaling model (MULTISIG1) was recently introduced to simulate the kinetics of repair of DNA double-strand breaks (DSBs) that were induced in confluent (non-dividing) cultured cells by a very low radiation dose where at most a single induced DSB would be expected in a given cell nucleus. The repair kinetics was modeled as representing what is now called an epigenetically-regulated (epiregulated) cell-community-wide (epicellcom) response to radiation stress. DSB repair initiation is assumed to require a threshold number of cells with DSBs participating in intercellular stress-response signaling. The MULTISIG1 model is extended in this study to apply to moderate doses where several DSBs can occur on the same DNA molecule. The repair of multiple breaks on the same molecule is treated as sequential stochastic events. For cells of differing genetic characteristics and epigenetic statuses, relationships are provided for evaluating the relative susceptibility (RS) for DSB induction, relative repair capacity (RRC) for DSB repair, and relative epiapoptosis capacity (REC), for epigenetically regulated apoptosis. The modified MULTISIG1 model is used to characterize the expected repair kinetics for confluent, human lung fibroblasts (MRC-5 line) briefly exposed in vitro to 90-kV x-rays. Possible application of the model to biological dosimetry is also discussed.

Feedback inhibition of L1 and alu retrotransposition through altered double strand break repair kinetics

BackgroundCells adapt to various chronic toxic exposures in a multitude of ways to minimize further damage and maximize their growth potential. Expression of L1 elements in the human genome can be greatly deleterious to cells, generating numerous double strand breaks (DSBs). Cells have been reported to respond to chronic DSBs by altering the repair of these breaks, including increasing the rate of homology independent DSB repair. Retrotransposition is strongly affected by proteins involved in DSB repair. Therefore, L1 expression has the potential to be a source of chronic DSBs and thus bring about the changes in cellular environment that could ultimately restrict its own retrotransposition.ResultsWe demonstrate that constitutive L1 expression leads to quicker DSB repair and decreases in the retrotransposition potential of L1 and other retrotransposons dependent on L1 expression for their mobility. This cellular adaptation results in reduced sensitivity to L1 induced toxicity. These effects can be induced by constitutive expression of the functional L1 ORF2 alone, but not by the constitutive expression of an L1 open reading frame 2 with mutations to its endonuclease and reverse transcriptase domains. This adaptation correlates with the relative activity of the L1 introduced into the cells.ConclusionsThe increased number of DSBs resulting from constitutive expression of L1 results in a more rapid rate of repair. The cellular response to this L1 expression also results in attenuation of retrotransposition and reduced sensitivity of the cells to negative consequences of L1 ORF2 expression. The influence does not appear to be through RNA interference. We believe that the increased rate of DSB repair is the most likely cause of the attenuation of retrotransposition. These alterations act as a fail safe mechanism that allows cells to escape the toxicity associated with the unchecked L1 expression. This gives cells that overexpress L1, such as tumor cells, the ability to survive the high levels of expression. However, the increased rate of break repair may come at the cost of accuracy of repair of the lesion, resulting in increased genomic instability.

Xrcc1-dependent and Ku-dependent DNA double-strand break repair kinetics in Arabidopsis plants

Double-strand breakage (DSB) of DNA involves loss of information on the two strands of the DNA fibre and thus cannot be repaired by simple copying of the complementary strand which is possible with single-strand DNA damage. Homologous recombination (HR) can precisely repair DSB using another copy of the genome as template and non-homologous recombination (NHR) permits repair of DSB with little or no dependence on DNA sequence homology. In addition to the well-characterised Ku-dependent non-homologous end-joining (NHEJ) pathway, much recent attention has been focused on Ku-independent NHR. The complex interrelationships and regulation of NHR pathways remain poorly understood, even more so in the case of plants, and we present here an analysis of Ku-dependent and Ku-independent repair of DSB in Arabidopsis thaliana. We have characterised an Arabidopsis xrcc1 mutant and developed quantitative analysis of the kinetics of appearance and loss of γ-H2AX foci as a tool to measure DSB repair in dividing root tip cells of γ-irradiated plants in vivo. This approach has permitted determination of DSB repair kinetics in planta following a short pulse of γ-irradiation, establishing the existence of a Ku-independent, Xrcc1-dependent DSB repair pathway. Furthermore, our data show a role for Ku80 during the first minutes post-irradiation and that Xrcc1 also plays such a role, but only in the absence of Ku. The importance of Xrcc1 is, however, clearly visible at later times in the presence of Ku, showing that alternative end-joining plays an important role in DSB repair even in the presence of active NHEJ.

Roles of Hop1 and Mek1 in meiotic chromosome pairing and recombination partner choice in Schizosaccharomyces pombe.

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.

Multicellular signalling model for DNA double-strand break repair kinetics after low-dose radiation

This paper introduces the Multicellular Signalling (MULTISIG1) model in which DNA double-strand break (DSB) repair initiation requires intercellular stress-response signalling from a threshold number of cells with DSBs, which occurs at the threshold radiation absorbed dose, T. The model applies to low radiation doses and employs an exponential distribution of times to DNA DSB repair completion with mean β. The MULTISIG1 model is found to be consistent with published data for γ-H2AX foci removal (reflecting DSB repair kinetics) after exposing cultures of confluent (non-dividing) human-derived lung fibroblast (MRC-5 line) to low-dose, 90-kV machine X-rays. T is estimated to be slightly greater than 1.2 mGy, a dose associated with, on average, about 1 electron track (form ionisations) per cell nucleus. β is estimated to be about 2.5 h and the corresponding rate of break repairs (μ) is estimated to be about 0.4 break repairs/h on different DNA molecules.

Competition effect in DNA damage response

We have built an ion-microbeam for studies of the nuclear topography and kinetics of double-strand break repair at the single cell level. Here, we show that a first and a second, delayed single ion exposure at different nuclear sites led to comparable accumulations of phospho-ATM, gamma-H2AX and Mdc1 at both earlier (e) and later (l) microirradiated sites. In contrast, accumulations of 53BP1 and the recombination protein Rad51 were strongly reduced at l-sites. This apparent competition effect is accompanied by a reduced amount of 53BP1 in undamaged areas of the irradiated nuclei. We suggest that a critically limited pool size combined with strong binding at irradiated sites leads to the exhaustion of unbound factors freely roaming the nuclear space. The undersupply of these factors at l-sites requires in addition a long-lasting binding at e-sites or a weaker binding at l-sites. The observed effects suggest that DNA damage response at individual nuclear sites depends on the time course of damage load. This may have implications for therapeutic radiation treatments.

Artemis is a phosphorylation target of ATM and ATR and is involved in the G2/M DNA damage checkpoint response.

Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemis-depleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.

The checkpoint protein Rad24 of Saccharomyces cerevisiae is involved in processing double-strand break ends and in recombination partner choice.

Upon chromosomal damage, cells activate a checkpoint response that includes cell cycle arrest and a stimulation of DNA repair. The checkpoint protein Rad24 is key to the survival of a single, repairable double-strand break (DSB). However, the low survival of rad24 cells is not due to their inability to arrest cell cycle progression. In rad24 mutants, processing of the broken ends is delayed and protracted, resulting in extended kinetics of DSB repair and in cell death. The limited resection of rad24 mutants also affects recombination partner choice by a mechanism dependent on the length of the interacting homologous donor sequences. Unexpectedly, rad24 cells with a DSB eventually accumulate and die at the G(2)/M phase of the cell cycle. This arrest depends on the spindle checkpoint protein Mad2.

Mechanisms of induction and repair of DNA double-strand breaks by ionizing radiation: some contradictions.

The various aspects of formation and repair of radiation-induced double-strand breaks (DSB) are summarized. Concerning the structure of DSB found in irradiated cells, enzymatic and microdosimetric analysis hints at complex damage of the DNA structure at the position of a DSB. With increasing LET, the DSB damage may be more complex than that induced by low-LET irradiation. Most of the DSB are repaired in the irradiated cell; apparently the kinetics of DSB repair and the fraction of unrejoined DSB determine cell survival or cell death. We do not know the details of the complex machinery of DSB repair; certainly recombination processes are involved, but there are still contradictions between our current knowledge about the mechanisms of recombinational DSB repair and the observed kinetics.

Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli.

To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI+ cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction.

Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae.

We studied the repair of double-strand breaks (DSB) in plasmid DNA introduced into haploid cells of the yeast Saccharomyces cerevisiae. The efficiency of repair was estimated from the frequency of transformation of the cells by an autonomously replicated linearized plasmid. The frequency of "lithium" transformation of Rad+ cells was increased greatly (by 1 order of magnitude and more) compared with that for circular DNA if the plasmid was initially linearized at the XhoI site within the LYS2 gene. This effect is due to recombinational repair of the plasmid DNA. Mutations rad52, rad53, rad54 and rad57 suppress the repair of DSB in plasmid DNA. The kinetics of DSB repair in plasmid DNA are biphasic: the first phase is completed within 1 h and the second within 14-18 h of incubating cells on selective medium.