A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double-strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild-type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.     

Pch2 modulates chromatid partner choice during meiotic double-strand break repair in Saccharomyces cerevisiae

In most organisms, the segregation of chromosomes during the first meiotic division is dependent upon at least one crossover (CO) between each pair of homologous chromosomes. COs can result from chromosome double-strand breaks (DSBs) that are induced and preferentially repaired using the homologous chromosome as a template. The PCH2 gene of budding yeast is required to establish proper meiotic chromosome axis structure and to regulate meiotic interhomolog DSB repair outcomes. These roles appear conserved in the mouse ortholog of PCH2, Trip13, which is also involved in meiotic chromosome axis organization and the regulation of DSB repair. Using a combination of genetic and physical assays to monitor meiotic DSB repair, we present data consistent with pch2Δ mutants showing defects in suppressing intersister DSB repair. These defects appear most pronounced in dmc1Δ mutants, which are defective for interhomolog repair, and explain the previously reported observation that pch2Δ dmc1Δ cells can complete meiosis. Results from genetic epistasis analyses involving spo13Δ, rad54Δ, and mek1/MEK1 alleles and an intersister recombination reporter assay are also consistent with Pch2 acting to limit intersister repair. We propose a model in which Pch2 is required to promote full Mek1 activity and thereby promotes interhomolog repair.     

Intra-chromosomal gene conversion induced by a DNA double-strand break in Saccharomyces cerevisiae

We have stimulated mitotic and meiotic gene conversion between non-tandem direct repeats of ADE4 by a defined double-strand break imparted in vivo to one of two copies of the gene. The experimental design permitted us to distinguish unambiguously between reciprocal intra-chromosomal crossing over and non-reciprocal break-join events that could accompany the induced conversions. We observed that (1) less than 10% of the induced conversion events are accompanied by intra-chromosomal crossing over in both mitosis and meiosis; (2) non-reciprocal break-join is not stimulated by the double-strand breaks; (3) a double-strand break in meiosis is repaired off intra-chromosomal homology (if available) with approximately sevenfold preference over repair off the homologous chromosome. Our observations, analyzed in the light of previous investigations of spontaneous inter and intra-chromosomal crossing over and gene conversion, lead to the view that chromosomal configuration constrains intra-chromosomal crossing over accompanying conversion between closely spaced repeated genes during resolution of the conversion intermediate.     

DNA strand breaks signal the induction of DNA double-strand break repair in Saccharomyces cerevisiae

Genotoxic stress induces a checkpoint signaling cascade to generate a stress response. Saccharomyces cerevisiae shows an altered radiation response under different type of stress. Although the induction of repair has been implicated in enhanced survival after exposure to the challenging stress, the nature of the signal remains poorly understood. This study demonstrates that low doses of gamma radiation and bleomycin induce RAD52-dependent recombination repair pathway in the wild-type strain D-261. Prior exposure of cells to DNA-damaging agents (gamma radiation or bleomycin) equips them better for the subsequent damage caused by challenging doses. However, exposure to UV light, which does not cause strand breaks, was ineffective. This was confirmed by PFGE studies. This indicates that the strand breaks probably serve as the signal for induction of the recombination repair pathway while pyrimidine dimers do not. The nature of the induced repair was investigated by mutation scoring in special strain D-7, which showed that the induced repair is essentially error free.     

Genome-wide redistribution of meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. DSBs are not randomly distributed along chromosomes. To better understand factors that control the distribution of DSBs in budding yeast, we have examined the genome-wide binding and cleavage properties of the Gal4 DNA binding domain (Gal4BD)-Spo11 fusion protein. We found that Gal4BD-Spo11 cleaves only a subset of its binding sites, indicating that the association of Spo11 with chromatin is not sufficient for DSB formation. In centromere-associated regions, the centromere itself prevents DSB cleavage by tethered Gal4BD-Spo11 since its displacement restores targeted DSB formation. In addition, we observed that new DSBs introduced by Gal4BD-Spo11 inhibit surrounding DSB formation over long distances (up to 60 kb), keeping constant the number of DSBs per chromosomal region. Together, these results demonstrate that the targeting of Spo11 to new chromosomal locations leads to both local stimulation and genome-wide redistribution of recombination initiation and that some chromosomal regions are inherently cold regardless of the presence of Spo11.     

Excess single-stranded DNA inhibits meiotic double-strand break repair

During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.     

Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae

DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.     

Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair.

DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I-IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae. Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian DNA ligase IV. Unlike CDC9, LIG4 is not essential for DNA replication, RAD52-dependent homologous recombination nor the repair of UV light-induced DNA damage. Instead, it encodes a crucial component of the non-homologous end-joining (NHEJ) apparatus, which repairs DNA double-strand breaks that are generated by ionizing radiation or restriction enzyme digestion: a function which cannot be complemented by CDC9. Lig4p acts in the same DNA repair pathway as the DNA end-binding protein Ku. However, unlike Ku, it does not function in telomere length homeostasis. These findings indicate diversification of function between different eukaryotic DNA ligases. Furthermore, they provide insights into mechanisms of DNA repair and suggest that the NHEJ pathway is highly conserved throughout the eukaryotic kingdom.     

Influence of non-homology between recombining DNA sequences on double-strand break repair in Saccharomyces cerevisiae

In this paper we study the influence of non-homology between plasmid and chromosomal DNA on the efficiency of recombinational repair of plasmid double-strand breaks and gaps in yeast. For this purpose we used different combinations of plasmids and yeast strains carrying various deletions within the yeast LYS2 gene. A 400 by deletion in plasmid DNA had no effect on recombinational plasmid repair. However, a 400 by deletion in chromosomal DNA dramatically reduced the efficiency of this repair mechanism, but recombinational repair of plasmids linearized by a double-strand break with cohesive ends still remained the dominant repair process. We have also studied the competition between recombination and ligation in the repair of linearized plasmids. Our experimental evidence suggests that recombinational repair is attempted but aborted if only one recombinogenic end with homology to chromosomal DNA is present in plasmid DNA. This situation results in a decreased probability of non-recombinational (i.e. ligation) repair of linearized plasmid DNA.     

DNA double-strand break repair within heterochromatic regions

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.     

Sae2p phosphorylation is crucial for cooperation with Mre11p for resection of DNA double-strand break ends during meiotic recombination in Saccharomyces cerevisiae

Meiotic recombination is initiated by the introduction of DNA double-strand breaks (DSBs) at recombination hotspots. DSB ends are resected to yield ssDNA, which is used in a homology search. Sae2p, which is involved in the resection of DSB ends, is phosphorylated by the Mec1p and Tel1p kinases during meiosis. To clarify the role of Sae2p phosphorylation in meiotic recombination, three mutants with alanine substitutions (at two putative Mec1/Tel1 phosphorylation sites near the N terminus, at three sites near the C terminus or at all five sites) were constructed. Analysis of DSB ends during meiotic recombination demonstrated that phosphorylation of the three C-terminal phosphorylation sites is necessary for DSB end resection and that phosphorylation of the two N-terminal phosphorylation sites is required for the efficient initiation of DSB end resection. Sae2p was localized on meiotic chromosomes in the rad50S and mre11-H125R mutants, which accumulate DSB ends. Alanine substitutions of all phosphorylation sites did not affect localization of Sae2p on meiotic chromosomes. Although colocalization of Sae2p with Mre11p and recombinant formation were observed in the N-terminally mutated and the C-terminally mutated strains, these processes were drastically impaired in the quintuple mutant. These results indicate that phosphorylation of Sae2p is required to initiate resection and to improve the efficiency of resection through cooperation with the Mre11-Rad50-Xrs2 complex.     

Aberrant double-strand break repair in rad51 mutants of Saccharomyces cerevisiae

A number of studies of Saccharomyces cerevisiae have revealed RAD51-independent recombination events. These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication. Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism. We propose a model for RAD51-independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing. The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required for RAD51-independent events by this model. Double-strand break repair between plasmid-borne inverted repeats was less efficient in rad1 rad51 double mutants than in rad1 and rad51 strains. In addition, repair events were delayed and frequently associated with plasmid loss. Furthermore, the repair products recovered from the rad1 rad51 strain were primarily in the crossover configuration, inconsistent with conservative models for mitotic double-strand break repair.     

Separable roles for Exonuclease I in meiotic DNA double-strand break repair

Exo1 is a member of the Rad2 protein family and possesses both 5'-3' exonuclease and 5' flap endonuclease activities. In addition to performing a variety of functions during mitotic growth, Exo1 is also important for the production of crossovers during meiosis. However, its precise molecular role has remained ambiguous and several models have been proposed to account for the crossover deficit observed in its absence. Here, we present physical evidence that the nuclease activity of Exo1 is essential for normal 5'-3' resection at the Spo11-dependent HIS4 hotspot in otherwise wild-type cells. This same activity was also required for normal levels of gene conversion at the locus. However, gene conversions were frequently observed at a distance beyond that at which resection was readily detectable arguing that it is not the extent of the initial DNA end resection that limits heteroduplex formation. In addition to these nuclease-dependent functions, we found that an exo1-D173A mutant defective in nuclease activity is able to maintain crossing-over at wild-type levels in a number of genetic intervals, suggesting that Exo1 also plays a nuclease-independent role in crossover promotion.     

Inhibition of DNA double-strand break repair by the Ku heterodimer in mrx mutants of Saccharomyces cerevisiae

Yeast rad50 and mre11 nuclease mutants are hypersensitive to physical and chemical agents that induce DNA double-strand breaks (DSBs). This sensitivity was suppressed by elevating intracellular levels of TLC1, the RNA subunit of telomerase. Suppression required proteins linked to homologous recombination, including Rad51, Rad52, Rad59 and Exo1, but not genes of the nonhomologous end-joining (NHEJ) repair pathway. Deletion mutagenesis experiments demonstrated that the 5'-end of TLC1 RNA was essential and a segment containing a binding site for the Yku70/Yku80 complex was sufficient for suppression. A mutant TLC1 RNA unable to associate with Yku80 protein did not increase resistance. These and other genetic studies indicated that association of the Ku heterodimer with broken DNA ends inhibits recombination in mrx mutants, but not in repair-proficient cells or in other DNA repair single mutants. In support of this model, DNA damage resistance of mrx cells was enhanced when YKU70 was co-inactivated. Defective recombinational repair of DSBs in mrx cells thus arises from at least two separate processes: loss of Mrx nuclease-associated DNA end-processing and inhibition of the Exo1-mediated secondary recombination pathway by Ku.     

Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in Saccharomyces cerevisiae

Nonhomologous end joining (NHEJ) is an important DNA double-strand-break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae: the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2), and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO-endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.     

Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae.

During repair of a double-strand break (DSB) by gene conversion, one or both 3' ends of the DSB invade a homologous donor sequence and initiate new DNA synthesis. The use of the invading DNA strand as a primer for new DNA synthesis requires that any nonhomologous bases at the 3' end be removed. We have previously shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3. We now report that these four proteins are needed only when the nonhomologous ends of recombining DNA are 30 nucleotides (nt) long or longer. An additional protein, the helicase Srs2, is required for the RAD1-dependent removal of long 3' tails. We suggest that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog. 3' tails shorter than 30 nt are removed by another mechanism that depends at least in part on the 3'-to-5' proofreading activity of DNA polymerase delta.     

Saccharomyces cerevisiae Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation

In budding yeast, at least 10 proteins are required for formation of the double-strand breaks (DSBs) that initiate meiotic recombination. Spo11 is the enzyme responsible for cleaving DNA and is found in a complex that also contains Ski8, Rec102, and Rec104. The Mre11/Rad50/Xrs2 complex is required for both DSB formation and DSB processing. In this article we investigate the functions of the remaining three proteins--Mer2, Mei4, and Rec114--with particular emphasis on Mer2. The Mer2 protein is present in vegetative cells, but it increases in abundance and becomes phosphorylated specifically during meiotic prophase. Mer2 localizes to distinct foci on meiotic chromosomes, with foci maximally abundant prior to the formation of synaptonemal complex. If DSB formation is blocked (e.g., by a spo11 mutation), dephosphorylation of Mer2 and its dissociation from chromosomes are delayed. We have also found that the Mei4 and Rec114 proteins localize to foci on chromosomes and these foci partially colocalize with each other and with Mer2. Furthermore, the three proteins co-immunoprecipitate. Mer2 does not show significant colocalization with Mre11 or Rec102 and Mer2 does not co-immunoprecipitate with Rec102. We propose that Mer2, Mei4, and Rec114 form a distinct complex required for DSB formation.