NEJ1 controls non-homologous end joining in Saccharomyces cerevisiae.

Broken DNA ends are rejoined by non-homologous end-joining (NHEJ) pathways requiring the Ku proteins (Ku70, Ku80), DNA ligase IV and its associated protein Lif1/Xrcc4 (ref. 1). In mammalian meiotic cells, Ku protein levels are much lower than in somatic cells, apparently reducing the capacity of meiotic cells to carry out NHEJ and thereby promoting homologous recombination. In Saccharomyces cerevisiae, NHEJ is also downregulated in meiosis-competent MATa/MAT alpha diploid cells in comparison with diploids or haploids expressing only MATa or MAT alpha. Diploids expressing both MATa and MAT alpha show enhanced mitotic homologous recombination. Here we report that mating-type-dependent regulation of NHEJ in budding yeast is caused in part by transcriptional repression of both LIF1 and the gene NEJ1 (YLR265C)--identified from microarray screening of messenger RNAs. Deleting NEJ1 reduces NHEJ 100-fold in MATa or MAT alpha haploids. Constitutive expression of NEJ1, but not expression of LIF1, restores NHEJ in MATa/MAT alpha cells. Nej1 regulates the subcellular distribution of Lif1. A green fluorescent protein (GFP)-Lif1 fusion protein accumulates in the nucleus in cells expressing NEJ1 but is largely cytoplasmic when NEJ1 is repressed.

The fuss about Mus81.

Endonucleolytic cleavage of Holliday junctions is important in recombination and replication. Mus81 proteins in yeasts and humans appear to have many, but not all, of the expected properties of eukaryotic Holliday junction resolvases, with intriguing connections to DNA replication checkpoints.

The Saccharomyces recombination protein Tid1p is required for adaptation from G2/M arrest induced by a double-strand break.

Saccharomyces cells with a single unrepaired double-strand break (DSB) will adapt to checkpoint-mediated G2/M arrest and resume cell cycle progression. The decision to adapt is finely regulated by the extent of single-stranded DNA generated from a DSB. We show that cells lacking the recombination protein Tid1p are unable to adapt, but that this defect is distinct from any role in recombination. As with the adaptation-defective mutations yku70Delta and cdc5-ad, permanent arrest in tid1Delta is bypassed by the deletion of the checkpoint gene RAD9. Permanent arrest of tid1Delta cells is suppressed by the rfa1-t11 mutation in the ssDNA binding complex RPA, similar to yku70Delta, whereas the defect in cdc5-ad is not suppressed. Unlike yku70Delta, tid1Delta does not affect 5'-to-3' degradation of DSB ends. The tid1Delta defect cannot be complemented by overexpressing the homolog Rad54p, nor is it affected in rad51Delta tid1Delta, rad54Delta tid1Delta, or rad52Delta tid1Delta double mutants that prevent essentially all homologous recombination. We suggest that Tid1p participates in monitoring the extent of single-stranded DNA produced by resection of DNA ends in a fashion that is distinct from its role in recombination.

Break-induced replication: a review and an example in budding yeast.

Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5' end of the LEU2 gene at one end was transformed into a strain in which the 5' end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2(+). Ade1(+) Leu2(+) transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.

A Saccharomyces servazzii clone homologous to Saccharomyces cerevisiae chromosome III spanning KAR4, ARS 304 and SPB1 lacks the recombination enhancer but contains an unknown ORF.

In order to learn about the evolutionary conservation of the recombination enhancer (RE) that controls donor preference during mating type switching in Saccharomyces cerevisiae, we have cloned a 13 kb region from S. servazzii. We find that the order of four genes surrounding the RE in S. cerevisiae (PRD1, KAR4, SPB1 and PBN1) is preserved in S.servazzii. However, there is an additional ORF in S. servazzii between PRD1 and KAR4 that is not homologous to any gene in S. cerevisiae or to genes in other organisms. Despite a 75-79% amino acid identity for KAR4 and SPB1, respectively, the S. servazzii sequence did not carry a well-conserved RE sequence and these sequences lacked RE function when introduced into S. cerevisiae. The S. servazzii region contains a sequence that supports autonomous DNA replication in S. cerevisiae and may represent a homologue of ARS304. The S. servazziii sequence has Genbank Accession No. BankIt359091 AF307954.

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.

RAD51-independent break-induced replication to repair a broken chromosome depends on a distant enhancer site.

Without the RAD51 strand exchange protein, Saccharomyces cerevisiae cannot repair a double-strand break (DSB) by gene conversion. However, cells can repair DSBs by recombination-dependent, break-induced replication (BIR). RAD51-independent BIR is initiated more than 13 kb from the DSB. Repair depends on a 200-bp sequence adjacent to ARS310, located approximately 34 kb centromere-proximal to the DSB, but does not depend on the origin activity of ARS310. We conclude that the ability of a recombination-induced replication fork to copy > 130 kb to the end of the chromosome depends on a special site that enhances assembly of a processive repair replication fork.

Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest.

Saccharomyces cells with one unrepaired double-strand break (DSB) adapt after checkpoint-mediated G2/M arrest. Adaptation is accompanied by loss of Rad53p checkpoint kinase activity and Chk1p phosphorylation. Rad53p kinase remains elevated in yku70delta and cdc5-ad cells that fail to adapt. Permanent G2/M arrest in cells with increased single-stranded DNA is suppressed by the rfa1-t11 mutation, but this RPA mutation does not suppress permanent arrest in cdc5-ad cells. Checkpoint kinase activation and inactivation can be followed in G2-arrested cells, but there is no kinase activation in G1-arrested cells. We conclude that activation of the checkpoint kinases in response to a single DNA break is cell cycle regulated and that adaptation is an active process by which these kinases are inactivated.

Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break.

Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence of RAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as with rad51Delta cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1 (RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Delta rad50Delta, rad51Delta rad59Delta, and rad54Delta tid1Delta. These results demonstrate that there is a RAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumably MRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.

HO endonuclease-induced recombination in yeast meiosis resembles Spo11-induced events.

In meiosis, gene conversions are accompanied by higher levels of crossing over than in mitotic cells. To determine whether the special properties of meiotic recombination can be attributed to the way in which Spo11p creates double-strand breaks (DSBs) at special hot spots in Saccharomyces cerevisiae, we expressed the site-specific HO endonuclease in meiotic cells. We could therefore compare HO-induced recombination in a well-defined region both in mitosis and meiosis, as well as compare Spo11p- and HO-induced meiotic events. HO-induced gene conversions in meiosis were accompanied by crossovers at the same high level (52%) as Spo11p-induced events. Moreover, HO-induced crossovers were reduced 3-fold by a msh4Delta mutation that similarly affects Spo11p-promoted events. In a spo11Delta diploid, where the only DSB is made by HO, crossing over was significantly higher (27%) than in mitotic cells (

The DNA damage checkpoint signal in budding yeast is nuclear limited.

The nature of the DNA damage-induced checkpoint signal that causes the arrest of cells prior to mitosis is unknown. To determine if this signal is transmitted through the cytoplasm or is confined to the nucleus, we created binucleate heterokaryon yeast cells in which one nucleus suffered an unrepairable double-strand break, and the second nucleus was undamaged. In most of these binucleate cells, the damaged nucleus arrested prior to spindle elongation, while the undamaged nucleus completed mitosis, even when the strength of the damage signal was increased. The arrest of the damaged nucleus was dependent upon the function of the RAD9 checkpoint gene. Thus, the DNA damage checkpoint causing G2/M arrest is regulated by a signal that is nuclear limited.

The Saccharomyces cerevisiae Msh2 mismatch repair protein localizes to recombination intermediates in vivo.

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.

DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair.

A DNA double-strand break (DSB) created by the HO endonuclease in Saccharomyces 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. Deltarad59 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.

Partners and pathwaysrepairing a double-strand break.

Double-strand chromosome breaks can arise in a number of ways, by ionizing radiation, by spontaneous chromosome breaks during DNA replication, or by the programmed action of endonucleases, such as in meiosis. Broken chromosomes can be repaired either by one of several homologous recombination mechanisms, or by a number of nonhomologous repair processes. Many of these pathways compete actively for the repair of a double-strand break. Which of these repair pathways is used appears to be regulated developmentally, genetically and during the cell cycle.

Recombination-induced CAG trinucleotide repeat expansions in yeast involve the MRE11-RAD50-XRS2 complex.

Recombination induced by double-strand breaks (DSBs) in yeast leads to a higher proportion of expansions to contractions than does replication-associated tract length changes. Expansions are apparently dependent on the property of the repeat array to form hairpins, since DSB repair of a CAA(87) repeat induces only contractions of the repeat sequence. DSB-repair efficiency is reduced by 40% when DNA synthesis must traverse a CAG(98) array, as compared with a CAA(87) array. These data indicate that repair- associated DNA synthesis is inhibited by secondary structures formed by CAG(98) and that these structures promote repeat expansions during DSB repair. Overexpression of Mre11p or Rad50p suppresses the inhibition of DSB repair by CAG(98) and significantly increases the average size of expansions found at the recipient locus. Both effects are dependent on the integrity of the Mre11p-Rad50p-Xrs2p complex. The Mre11 complex thus appears to be directly involved in removing CAG or CTG hairpins that arise frequently during DNA synthesis accompanying gene conversion of these trinucleotide repeats.

Recombination: a frank view of exchanges and vice versa.

The study of double-strand chromosome break repair by homologous and nonhomologous recombination is a growth industry. In the past year, there have been important advances both in understanding the connection between recombination and DNA replication and in linking recombination with origins of human cancer. At the same time, a combination of biochemical, genetic, molecular biological, and cytological approaches have provided a clearer vision of the specific functions of a variety of recombination proteins.

Separation-of-function mutations in Saccharomyces cerevisiae MSH2 that confer mismatch repair defects but do not affect nonhomologous-tail removal during recombination.

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 MSH3 gene 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 msh2 mutations 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.