Association of Mre11p with double-strand break sites during yeast meiosis

The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex. In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination. We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome. Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50. However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F. Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs. We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.     

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.     

Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).     

Expanded CAG/CTG repeat DNA induces a checkpoint response that impacts cell proliferation in Saccharomyces cerevisiae

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases.     

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG)(n)/(CTG)(n) hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG)(n)/(CTG)(n) hairpin structures during DNA metabolism.     

Contractions and expansions of CAG/CTG trinucleotide repeats occur during ectopic gene conversion in yeast,by a MUS81-independent mechanism

CAG/CTG trinucleotide repeat tracts expand and contract at a high rate during gene conversion in Saccharomyces cerevisiae. In order to characterize the mechanism responsible for such rearrangements, we built an experimental system based on the use of the rare cutter endonuclease I-SceI, to study the fate of trinucleotide repeat tracts during meiotic or mitotic (allelic or ectopic) gene conversion. After double-strand break (DSB) induced meiotic recombination, (CAG)(98) and (CAG)(255) are rearranged in 5% and 52% of the gene conversions, respectively, with similar proportions of contractions and expansions. No evidence of a meiotic hot spot activity associated with trinucleotide repeats could be found. When gene conversion is induced by a DSB during mitotic growth of the cells, no rearrangement of the repeat tracts is detected when the donor sequence is allelic to the recipient site of the DSB. However, when the donor sequence is at an ectopic location, frequent contractions and expansions of the repeat tract are found. No crossing-over associated with gene conversion could be detected. Mutants for the MUS81 gene, involved in the resolution of recombination intermediates, show a frequency of rearrangements identical with that of the wild-type strain. We concluded that trinucleotide repeat rearrangements occur frequently during ectopic but not during allelic recombination, by a mechanism that does not require crossover formation.     

Arsenic-induced Mre11 phosphorylation is cell cycle-dependent and defective in NBS cells

Cancer-prone diseases ataxia-telangiectasia (AT), Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD) are defective in the repair of DNA double-stranded break (DSB). On the other hand, arsenic (As) has been reported to cause DSB and to be involved in the occurrence of skin, lung and bladder cancers. To dissect the repair mechanism of As-induced DSB, wild type, AT and NBS cells were treated with sodium arsenite to study the complex formation and post-translational modification of Rad50/NBS1/Mre11 repair proteins. Our results showed that Mre11 went through cell cycle-dependent phosphorylation upon sodium arsenite treatment and this post-translational modification required NBS1 but not ATM. Defective As-induced Mre11 phosphorylation was rescued by reconstitution with full length NBS1 in NBS cells. Although As-induced Mre11 phosphorylation was not required for Rad50/NBS1/Mre11 complex formation, it might be required for the formation of Rad50/NBS1/Mre11 nuclear foci upon DNA damage.     

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation

High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. ⁵⁶iron and ²⁸silicon) by inhibiting Mre11 complex activity. Exposure of cells to ⁵⁶iron or ²⁸silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.     

DNA double-strand breaks induce deletion of CTG.CAG repeats in an orientation-dependent manner in Escherichia coli

The influences of double-strand breaks (DSBs) within a triplet repeat sequence on its genetic instabilities (expansions and deletions) related to hereditary neurological diseases was investigated. Plasmids containing 43 or 70 CTG.CAG repeats or 43 CGG.CCG repeats were linearized in vitro near the center of the repeats and were transformed into parental, RecA-dependent homologous recombination-deficient, or RecBC exonuclease-deficient Escherichia coli. The resulting repair process considerably increased deletion of the repeating sequence compared to the circular DNA controls. Unexpectedly, the orientation of the insert relative to the unidirectional ColE1 origin of replication affected the amount of instability generated during the repair of the DSB. When the CTG strand was the template for lagging-strand synthesis, instability was increased, most markedly in the recA- strain. Results indicated that RecA and/or RecBC might play a role in DSB repair within the triplet repeat. Altering the length, orientation, and sequence composition of the triplet repeat suggested an important role of DNA secondary structures during repair intermediates. Hence, we hypothesize that ColE1 origin-dependent replication was involved during the repair of the DSB. A model is presented to explain the mechanisms of the observed genetic instabilities.     

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5′ ends of a bridged DSB are juxtaposed such that DNA unwinding and 3′–5′ exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5′ termini and exonucleolytic degradation of the 3′ ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.Key words: ATM, Mre11, MRN complex, DNA degradation, double-strand break repair, microhomology-mediated end joining, PI-3-kinase-like kinases     

Functional interactions between Sae2 and the Mre11 complex

The Mre11 complex functions in double-strand break (DSB) repair, meiotic recombination, and DNA damage checkpoint pathways. Sae2 deficiency has opposing effects on the Mre11 complex. On one hand, it appears to impair Mre11 nuclease function in DNA repair and meiotic DSB processing, and on the other, Sae2 deficiency activates Mre11-complex-dependent DNA-damage-signaling via the Tel1-Mre11 complex (TM) pathway. We demonstrate that SAE2 overexpression blocks the TM pathway, suggesting that Sae2 antagonizes Mre11-complex checkpoint functions. To understand how Sae2 regulates the Mre11 complex, we screened for sae2 alleles that behaved as the null with respect to Mre11-complex checkpoint functions, but left nuclease function intact. Phenotypic characterization of these sae2 alleles suggests that Sae2 functions as a multimer and influences the substrate specificity of the Mre11 nuclease. We show that Sae2 oligomerizes independently of DNA damage and that oligomerization is required for its regulatory influence on the Mre11 nuclease and checkpoint functions.     

Expansions and contractions in 36-bp minisatellites by gene conversion in yeast

The instability of simple tandem repeats, such as human minisatellite loci, has been suggested to arise by gene conversions. In Saccharomyces cerevisiae, a double-strand break (DSB) was created by the HO endonuclease so that DNA polymerases associated with gap repair must traverse an artificial minisatellite of perfect 36-bp repeats or a yeast Y' minisatellite containing diverged 36-bp repeats. Gene conversions are frequently accompanied by changes in repeat number when the template contains perfect repeats. When the ends of the DSB have nonhomologous tails of 47 and 70 nucleotides that must be removed before repair DNA synthesis can begin, 16% of gene conversions had rearrangements, most of which were contractions, almost always in the recipient locus. When efficient removal of nonhomologous tails was prevented in rad1 and msh2 strains, repair was reduced 10-fold, but among survivors there was a 10-fold reduction in contractions. Half the remaining events were expansions. A similar decrease in the contraction rate was observed when the template was modified so that DSB ends were homologous to the template; and here, too, half of the remaining rearrangements were expansions. In this case, efficient repair does not require RAD1 and MSH2, consistent with our previous observations. In addition, without nonhomologous DSB ends, msh2 and rad1 mutations did not affect the frequency or the distribution of rearrangements. We conclude that the presence of nonhomologous ends alters the mechanism of DSB repair, likely through early recruitment of repair proteins including Msh2p and Rad1p, resulting in more frequent contractions of repeated sequences.     

DNA damage-dependent nuclear dynamics of the Mre11 complex

The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage.     

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell-cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5' ends of a bridged DSB are juxtaposed such that DNA unwinding and 3'-5' exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5' termini and exonucleolytic degradation of the 3' ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.     

MSH2 ATPase Domain Mutation Affects CTG?CAG Repeat Instability in Transgenic Mice

Myotonic dystrophy type 1 (DM1) is associated with one of the most highly unstable CTG•CAG repeat expansions. The formation of further repeat expansions in transgenic mice carrying expanded CTG•CAG tracts requires the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSβ complex. It has been proposed that binding of MutSβ to CAG hairpins blocks its ATPase activity compromising hairpin repair, thereby causing expansions. This would suggest that binding, but not ATP hydrolysis, by MutSβ is critical for trinucleotide expansions. However, it is unknown if the MSH2 ATPase activity is dispensible for instability. To get insight into the mechanism by which MSH2 generates trinucleotide expansions, we crossed DM1 transgenic mice carrying a highly unstable >(CTG)₃₀₀ repeat tract with mice carrying the G674A mutation in the MSH2 ATPase domain. This mutation impairs MSH2 ATPase activity and ablates base–base MMR, but does not affect the ability of MSH2 (associated with MSH6) to bind DNA mismatches. We found that the ATPase domain mutation of MSH2 strongly affects the formation of CTG expansions and leads instead to transmitted contractions, similar to a Msh2-null or Msh3-null deficiency. While a decrease in MSH2 protein level was observed in tissues from Msh2^G674 mice, the dramatic reduction of expansions suggests that the expansion-biased trinucleotide repeat instability requires a functional MSH2 ATPase domain and probably a functional MMR system.     

MSH2 ATPase Domain Mutation Affects CTG•CAG Repeat Instability in Transgenic Mice

Myotonic dystrophy type 1 (DM1) is associated with one of the most highly unstable CTG•CAG repeat expansions. The formation of further repeat expansions in transgenic mice carrying expanded CTG•CAG tracts requires the mismatch repair (MMR) proteins MSH2 and MSH3, forming the MutSβ complex. It has been proposed that binding of MutSβ to CAG hairpins blocks its ATPase activity compromising hairpin repair, thereby causing expansions. This would suggest that binding, but not ATP hydrolysis, by MutSβ is critical for trinucleotide expansions. However, it is unknown if the MSH2 ATPase activity is dispensible for instability. To get insight into the mechanism by which MSH2 generates trinucleotide expansions, we crossed DM1 transgenic mice carrying a highly unstable >(CTG)₃₀₀ repeat tract with mice carrying the G674A mutation in the MSH2 ATPase domain. This mutation impairs MSH2 ATPase activity and ablates base–base MMR, but does not affect the ability of MSH2 (associated with MSH6) to bind DNA mismatches. We found that the ATPase domain mutation of MSH2 strongly affects the formation of CTG expansions and leads instead to transmitted contractions, similar to a Msh2-null or Msh3-null deficiency. While a decrease in MSH2 protein level was observed in tissues from Msh2^G674 mice, the dramatic reduction of expansions suggests that the expansion-biased trinucleotide repeat instability requires a functional MSH2 ATPase domain and probably a functional MMR system.     

Nuclease-deficient FEN-1 blocks Rad51/BRCA1-mediated repair and causes trinucleotide repeat instability

Previous studies have shown that expansion-prone repeats form structures that inhibit human flap endonuclease (FEN-1). We report here that faulty processing by FEN-1 initiates repeat instability in mammalian cells. Disease-length CAG tracts in Huntington's disease mice heterozygous for FEN-1 display a tendency toward expansions over contractions during intergenerational inheritance compared to those in homozygous wild-type mice. Further, with regard to human cells expressing a nuclease-defective FEN-1, we provide direct evidence that an unprocessed FEN-1 substrate is a precursor to instability. In cells with no endogenous defects in DNA repair, exogenous nuclease-defective FEN-1 causes repeat instability and aberrant DNA repair. Inefficient flap processing blocks the formation of Rad51/BRCA1 complexes but invokes repair by other pathways.     

Most meiotic CAG repeat tract-length alterations in yeast are<Emphasis Type="Italic"> SPO11</Emphasis> dependent

The expansion of trinucleotide repeat sequences associated with hereditary neurological diseases is believed from earlier studies to be due to errors in DNA replication. However, more recent studies have indicated that recombination may play a significant role in triplet repeat expansion. CAG repeat tracts have been shown to induce double-strand breaks (DSBs) during meiosis in yeast, and DSB formation is dependent on the meiotic recombination machinery. The rate of meiotic instability is several fold higher than mitotic instability. To determine whether DSB repair is responsible for the high rate of repeat tract-length alterations, the frequencies of meiotic repeat-tract instability were compared in wild-type and spo11 mutant strains. In the spo11 background, the rate of meiotic repeat-tract instability remained at the mitotic level, suggesting that meiotic alterations of CAG repeat tracts in yeast occur by the recombination mechanism. Several of these meiotic tract-length alterations are due to DSB repair involving use of the sister chromatid as a template.     

Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1

Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.