[Show abstract][Hide abstract] ABSTRACT: Fun30 is a Swi2/Snf2 homolog in budding yeast that has been shown to remodel chromatin both in vitro and in vivo. We report that Fun30 plays a key role in homologous recombination, by facilitating 5′-to-3′ resection of double-strand break
(DSB) ends, apparently by facilitating exonuclease digestion of nucleosome-bound DNA adjacent to the DSB. Fun30 is recruited
to an HO endonuclease-induced DSB and acts in both the Exo1-dependent and Sgs1-dependent resection pathways. Deletion of FUN30 slows the rate of 5′-to-3′ resection from 4 kb/h to about 1.2 kb/h. We also found that the resection rate is reduced by DNA
damage-induced phosphorylation of histone H2A-S129 (γ-H2AX) and that Fun30 interacts preferentially with nucleosomes in which
H2A-S129 is not phosphorylated. Fun30 is not required for later steps in homologous recombination. Like its homolog Rdh54/Tid1,
Fun30 is required to allow the adaptation of DNA damage checkpoint-arrested cells with an unrepaired DSB to resume cell cycle
[Show abstract][Hide abstract] ABSTRACT: DNA double-strand breaks (DSBs) have proven to be very potent initiators of recombination in yeast and other organisms. A single, site-specific DSB initiates homologous DNA repair events such as gene conversion, break-induced replication, and single-strand annealing, as well as nonhomologous end joining, microhomology-mediated end joining, and new telomere addition. When repair is either delayed or prevented, a single DSB can trigger checkpoint-mediated cell cycle arrest. In budding yeast, expressing the HO endonuclease under the control of a galactose-inducible promoter has been instrumental in the study of these processes by providing us a way to synchronously induce a DSB at a unique site in vivo. We describe how the HO endonuclease has been used to study the recombination events in mating-type (MAT) switching. Southern blots provide an overview of the process by allowing one to examine the formation of the DSB, DNA degradation at the break, and formation of the product. Denaturing gels and slot blots as well as PCR have provided important tools to follow the progression of resection in wild-type and mutant cells. PCR has also been important in allowing us to follow the kinetics of certain recombination intermediates such as the initiation of repair DNA synthesis or the removal of nonhomologous Y sequences during MAT switching. Finally chromatin immunoprecipitation has been used to follow the recruitment of key proteins to the DSB and in subsequent steps in DSB repair.
No preview · Article · Sep 2012 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Budding yeast Slx4 interacts with the Rad1-Rad10 endonuclease that is involved in nucleotide excision repair (NER), homologous recombination (HR) and single-strand annealing (SSA). We previously showed that Slx4 is dispensable for NER but is essential for SSA. Slx4 is phosphorylated by the Mec1 and Tel1 kinases after DNA damage on at least six Ser/Thr residues, and mutation of all six residues to Ala reduces the efficiency of SSA. In this study, we further investigated the role of Slx4 phosphorylation in SSA, specifically in regulating cleavage of 3' non-homologous (NH) DNA tails by Rad1-Rad10 during SSA and HR. Slx4 became phosphorylated after induction of a single double-strand break (DSB) during SSA and dephosphorylation coincided approximately with completion of repair. Slx4 is recruited to 3' NH tails during DSB repair, but this does not require phosphorylation of Slx4. However, we identified a specific damage-dependent Mec1/Tel1 site of Slx4 phosphorylation, Thr 113, that is required for efficient cleavage of NH tails by Rad1-Rad10. Consistent with these data, deletion of both Mec1 and Tel1 severely reduces the efficiency of NH DNA tail cleavage during HR. These data show that phosphorylation of Slx4 by Mec1 and Tel1 plays an important role in facilitating NH DNA tail cleavage during HR.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cells employ a suite of replication and mitotic checkpoints to ensure the accurate transmission of their DNA. In budding yeast, both the DNA damage checkpoint and the spindle assembly checkpoint (SAC) block cells prior to anaphase. The presence of a single unrepaired double-strand break (DSB) activates ATR and ATM protein kinase homologs Mec1 and Tel1, which then activate downstream effectors to trigger G2/M arrest and also phosphorylate histone H2A (creating gamma-H2AX) in chromatin surrounding the DSB. The SAC monitors proper attachment of spindle microtubules to the kinetochore formed at each centromere and the biorientation of sister centromeres toward opposite spindle pole bodies. Although these two checkpoints sense quite different perturbations, recent evidence has demonstrated both synergistic interactions and cross-talk between them. Here we report that Mad2 and other SAC proteins play an unexpected role in prolonging G2/M arrest after induction of a single DSB. This function of the SAC depends not only on Mec1 and other components of the DNA damage checkpoint but also on the presence of the centromere located > or = 90 kb from the DNA damage. DNA damage induces epigenetic changes at the centromere, including the gamma-H2AX modification, that appear to alter kinetochore function, thus triggering the canonical SAC. Thus, a single DSB triggers a response by both checkpoints to prevent the segregation of a damaged chromosome.
Full-text · Article · Feb 2010 · Current biology: CB
[Show abstract][Hide abstract] ABSTRACT: A DNA double-strand break (DSB) is repaired by gene conversion (GC) if both ends of the DSB share homology with an intact DNA sequence. However, if homology is limited to only one of the DSB ends, repair occurs by break-induced replication (BIR). It is not known how the homology status of the DSB ends is first assessed and what other parameters govern the choice between these repair pathways. Our data suggest that a "recombination execution checkpoint" (REC) regulates the choice of the homologous recombination pathway employed to repair a given DSB. This choice is made prior to the initiation of DNA synthesis, and is dependent on the relative position and orientation of the homologous sequences used for repair. The RecQ family helicase Sgs1 plays a key role in regulating the choice of the recombination pathway. Surprisingly, break repair and gap repair are fundamentally different processes, both kinetically and genetically, as Pol32 is required only for gap repair. We propose that the REC may have evolved to preserve genome integrity by promoting conservative repair, especially when a DSB occurs within a repeated sequence.
Full-text · Article · Mar 2009 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: Budding yeast (Saccharomyces cerevisiae) Slx4 is essential for cell viability in the absence of the Sgs1 helicase and for recovery from DNA damage. Here we report
that cells lacking Slx4 have difficulties in completing DNA synthesis during recovery from replisome stalling induced by the
DNA alkylating agent methyl methanesulfonate (MMS). Although DNA synthesis restarts during recovery, cells are left with unreplicated
gaps in the genome despite an increase in translesion synthesis. In this light, epistasis experiments show that SLX4 interacts with genes involved in error-free bypass of DNA lesions. Slx4 associates physically, in a mutually exclusive manner,
with two structure-specific endonucleases, Rad1 and Slx1, but neither of these enzymes is required for Slx4 to promote resistance
to MMS. However, Rad1-dependent DNA repair by single-strand annealing (SSA) requires Slx4. Strikingly, phosphorylation of
Slx4 by the Mec1 and Tel1 kinases appears to be essential for SSA but not for cell viability in the absence of Sgs1 or for
cellular resistance to MMS. These results indicate that Slx4 has multiple functions in responding to DNA damage and that a
subset of these are regulated by Mec1/Tel1-dependent phosphorylation.
Full-text · Article · Oct 2007 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Double strand breaks (DSBs) can cause damage to the genomic integrity of a cell as well as initiate genetic recombination processes. The HO and I-SceI endonucleases from budding yeast have provided a way to study these events by inducing a unique DSB in vivo under the control of a galactose-inducible promoter. The GAL::HO construct has been used extensively to study processes such as nonhomologous end joining, intra- and interchromosomal gene conversion, single strand annealing and break-induced recombination. Synchronously induced DSBs have also been important in the study of the DNA damage checkpoint, adaptation, and recovery pathways of yeast. This chapter describes methods of using GAL::HO to physically monitor the progression of events following a DSB, specifically the events leading to the switching of mating type by gene conversion of MAT using the silent donors at HML and HMR. Southern blot analysis can be used to follow the overall events in this process such as the formation of the DSB and product. Denaturing alkaline gels and slot blot techniques can be employed to follow the 5' to 3' resection of DNA starting at the DSB. After resection, the 3' tail initiates a homology search and then strand invades its homologous sequence at the donor cassette. Polymerase chain reaction is an important means to assay strand invasion and the priming of new DNA synthesis as well as the completion of gene conversion. Methods such as chromatin immunoprecipitation have provided a means to study many proteins that associate with a DSB, including not only recombination proteins, but also proteins involved in nonhomologous end joining, cell cycle arrest, chromatin remodeling, cohesin function, and mismatch repair.
No preview · Article · Feb 2006 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta mlh3 Delta triple mutant resembled mlh1 Delta. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.
Full-text · Article · Jul 2004 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.
Full-text · Article · Feb 2004 · Philosophical Transactions of The Royal Society B Biological Sciences
[Show abstract][Hide abstract] ABSTRACT: Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G2/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p
fail to adapt, but deleting RAD52 suppresses rad51Δ. rad52Δ also suppresses adaptation defects of srs2Δ mutants but not those of yku70Δ or tid1Δ mutants. Neither rad54Δ nor rad55Δ affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal
truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA),
supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Δ mutants and fully restores adaptation in yku70Δ and tid1Δ mutants. Surprisingly, although neither rfa1-t11 nor rad52Δ mutants are adaptation defective, the rad52Δ rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53
after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p
to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.
Full-text · Article · Jan 2004 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Repairing a double-strand break by homologous recombination requires binding of the strand exchange protein Rad51p to ssDNA, followed by synapsis with a homologous donor. Here we used chromatin immunoprecipitation to monitor the in vivo association of Saccharomyces cerevisiae Rad51p with both the cleaved MATa locus and the HML alpha donor. Localization of Rad51p to MAT precedes its association with HML, providing evidence of the time needed for the Rad51 filament to search the genome for a homologous sequence. Rad51p binding to ssDNA requires Rad52p. The absence of Rad55p delays Rad51p binding to ssDNA and prevents strand invasion and localization of Rad51p to HML alpha. Lack of Rad54p does not significantly impair Rad51p recruitment to MAT or its initial association with HML alpha; however, Rad54p is required at or before the initiation of DNA synthesis after synapsis has occurred at the 3' end of the invading strand.
[Show abstract][Hide abstract] ABSTRACT: A DNA double-strand break (DSB) created by the HO endonuclease inSaccharomyces cerevisiae will stimulate recombination between flanking repeats by the single-strand annealing (SSA) pathway, producing a deletion.
Previously the efficiency of SSA, using homologous sequences of different lengths, was measured in competition with that of
a larger repeat further from the DSB, which ensured that nearly all cells would survive the DSB if the smaller region was
not used (N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12:563–575, 1992). Without competition, the efficiency with which
homologous segments of 63 to 205 bp engaged in SSA was significantly increased. A sequence as small as 29 bp was used 0.2%
of the time, and homology dependence was approximately linear up to 415 bp, at which size almost all cells survived. A mutant
with a deletion of RAD59, a homologue of RAD52, was defective for SSA, especially when the homologous-sequence length was short; however, even with 1.17-kb substrates,
SSA was reduced fourfold. DSB-induced gene conversion also showed a partial dependence on Rad59p, again being greatest when
the homologous-sequence length was short. We found that Rad59p plays a role in removing nonhomologous sequences from the ends
of single-stranded DNA when it invades a homologous DNA template, in a manner similar to that previously seen with srs2 mutants. Δrad59 affected DSB-induced gene conversion differently from msh3 and msh2, which are also defective in removing nonhomologous ends in both DSB-induced gene conversion and SSA. A msh3 rad59 double mutant was more severely defective in SSA than either single mutant.
Full-text · Article · Aug 2000 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Mismatch repair proteins act during double-strand break repair (DSBR) to correct mismatches in heteroduplex DNA, to suppress recombination between divergent sequences, and to promote removal of nonhomologous DNA at DSB ends. We investigated yeast Msh2p association with recombination intermediates in vivo using chromatin immunoprecipitation. During DSBR involving nonhomologous ends, Msh2p localized strongly to recipient and donor sequences. Localization required Msh3p and was greatly reduced in rad50delta strains. Minimal localization of Msh2p was observed during fully homologous repair, but this was increased in rad52delta strains. These findings argue that Msh2p-Msh3p associates with intermediates early in DSBR to participate in the rejection of homeologous pairing and to stabilize nonhomologous tails for cleavage by Rad1p-Rad10p endonuclease.
[Show abstract][Hide abstract] ABSTRACT: Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to
their role in mismatch repair (MMR), the MSH2 and MSH3gene products are required to remove 3′ nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6,MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped
to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the
removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein
complexes that are thought to play different roles during DNA repair and genetic recombination.
Full-text · Article · Dec 1999 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.
[Show abstract][Hide abstract] ABSTRACT: When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3' tail.
Full-text · Article · Sep 1997 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with > 80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching.
[Show abstract][Hide abstract] ABSTRACT: In Saccharomyces cerevisiae, HO endonuclease-induced mating-type (MAT) switching is a specialized mitotic recombination event in which MAT sequences are replaced by those copied from a distant, unexpressed donor (HML or HMR). The donors have a chromatin structure inaccessible for both transcription and HO cleavage. Here we use physical monitoring of DNA to show that MAT switching is completely blocked at an early step in recombination in strains deleted for the DNA repair genes RAD51, RAD52, RAD54, RAD55 or RAD57. We find, however, that only RAD52 is required when the donor sequence is simultaneously not silenced and located on a plasmid. RAD51, RAD54, RAD55 and RAD57 are still required when the same transcribed donor is on the chromosome. We conclude that recombination in vivo occurs between DNA molecules in chromatin, whose structure significantly influences the outcome. RAD51, RAD54, RAD55 and RAD57 are all required to facilitate strand invasion into otherwise inaccessible donor sequences.
[Show abstract][Hide abstract] ABSTRACT: In Saccharomyces cerevisiae, a large number of genes in the RAD52 epistasis group has been implicated in the repair of chromosomal
double-strand breaks and in both mitotic and meiotic homologous recombination. While most of these genes are essential for
yeast mating-type (MAT) gene switching, neither RAD50 nor XRS2 is required to complete this specialized mitotic gene conversion
process. Using a galactose-inducible HO endonuclease gene to initiate MAT switching, we have examined the effect of null mutations
of RAD50 and of XRS2 on intermediate steps of this recombination event. Both rad50 and xrs2 mutants exhibit a marked delay
in the completion of switching. Both mutations reduce the extent of 5'-to-3' degradation from the end of the HO-created double-strand
break. The steps of initial strand invasion and new DNA synthesis are delayed by approximately 30 min in mutant cells. However,
later events are still further delayed, suggesting that XRS2 and RAD50 affect more than one step in the process. In the rad50
xrs2 double mutant, the completion of MAT switching is delayed more than in either single mutant, without reducing the overall
efficiency of the process. The XRS2 gene encodes an 854-amino-acid protein with no obvious similarity to the Rad50 protein
or to any other protein in the database. Overexpression of RAD50 does not complement the defects in xrs2 or vice versa.
Full-text · Article · Jun 1994 · Molecular and Cellular Biology