Homologous Recombination and Repair Functions Required for Mutagenicity during Yeast Meiosis.

Several meiotic events reshape the genome prior to its transfer (via gametes) to the next generation. The occurrence of new meiotic mutations is tightly linked to homologous recombination (HR) and firmly depends on Spo11-induced DNA breaks. To gain insight into the molecular mechanisms governing mutagenicity during meiosis, we examined the timing of mutation and recombination events in cells deficient in various DNA HR-repair genes, which represent distinct functions along the meiotic recombination process. Despite sequence similarities and overlapping activities of the two DNA translocases, Rad54 and Tid1, we observed essential differences in their roles in meiotic mutation occurrence: in the absence of Rad54, meiotic mutagenicity was elevated 8-fold compared to the wild type (WT), while in the tid1Δ mutant, there were few meiotic mutations, nine percent compared to the WT. We propose that the presence of Rad54 channels recombinational repair to a less mutagenic pathway, whereas repair assisted by Tid1 is more mutagenic. A 3.5-fold increase in mutation level was observed in dmc1∆ cells, suggesting that single-stranded DNA (ssDNA) may be a potential source for mutagenicity during meiosis. Taken together, we suggest that the introduction of de novo mutations also contributes to the diversification role of meiotic recombination. These rare meiotic mutations revise genomic sequences and may contribute to long-term evolutionary changes.

Mutagenicity in haploid yeast meiosis resulting from repair of DSBs by the sister chromatid.

Mutations in diploid budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. Such meiotic mutations are thought to result from the repair of double-strand breaks (DSBs) in meiosis, during the process of recombination. Here, we report studies of mutagenicity in haploid strains that may undergo meiosis due to the expression of both mating-type alleles, MATa and MATα. We measure the rate of mutagenicity in the reporter gene CAN1, and find it to be fivefold higher than in mitotic cells, as determined by fluctuation analysis. This enhanced meiotic mutagenicity is shown to depend on the presence of SPO11, the gene responsible for meiotic DSBs. Mutations in haploid meiosis must result from repair of the DSBs through interaction with the sister chromatid, rather than with non-sister chromatids as in diploids. Thus, mutations in diploid meiosis that are not ostensibly associated with recombination events can be explained by sister-chromatid repair. The spectrum of meiotic mutations revealed by Sanger sequencing is similar in haploid and in diploid meiosis. Compared to mitotic mutations in CAN1, long Indels are more frequent among meiotic mutations. Both, meiotic and mitotic mutations are more common at G/C sites than at A/T, in spite of an opposite bias in the target reporter gene. We conclude that sister-chromatid repair of DSBs is a major source of mutagenicity in meiosis.

Timing of appearance of new mutations during yeast meiosis and their association with recombination.

Mutations in budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. In contrast to mutations that occur in somatic cells, meiotic mutations have a special, long-range impact on evolution, because they are transferred to the following generations through the gametes. Understanding the mechanistic basis of meiotic mutagenicity is still lacking, however. Here, we report studies of mutagenicity in the reporter gene CAN1, in which forward mutation events in meiosis are sevenfold higher than in mitotic cells, as determined by fluctuation analysis. Meiotic mutations appear approximately at the same time as heteroallelic-recombination products and as meiotic DSBs. Recombination-associated timing of meiotic mutagenicity is further augmented by the absence of meiotic mutations in cells arrested after pre-meiotic DNA synthesis. More than 40% of the mutations generated in meiosis in CAN1 are found on chromosomes that have recombined in the 2.2 kb covering the reporter, implying that the mutations have resulted from recombination events and that meiotic recombination is mutagenic. The induced expression in yeast meiosis of low-fidelity DNA polymerases coded by the genes REV1, REV3, RAD30, and POL4 makes them attractive candidates for introducing mutations. However, in our extensive experiments with polymerase-deleted strains, these polymerases do not appear to be the major source of meiotic mutagenicity. From the connection between meiotic mutagenicity and recombination, one may conclude that meiotic recombination has another diversification role, of introducing new mutations at the DNA sequence level, in addition to reshuffling of existing variation. The new, rare meiotic mutations may contribute to long-range evolutionary processes and enhance adaptation to challenging environments.

Elevated Mutagenicity in Meiosis and Its Mechanism.

Diploid germ cells produce haploid gametes through meiosis, a unique type of cell division. Independent reassortment of parental chromosomes and their recombination leads to ample genetic variability among the gametes. Importantly, new mutations also occur during meiosis, at frequencies much higher than during the mitotic cell cycles. These meiotic mutations are associated with genetic recombination and depend on double-strand breaks (DSBs) that initiate crossing over. Indeed, sequence variation among related strains is greater around recombination hotspots than elsewhere in the genome, presumably resulting from recombination-associated mutations. Significantly, enhanced mutagenicity in meiosis may lead to faster divergence during evolution, as germ-line mutations are the ones that are transmitted to the progeny and thus have an evolutionary impact. The molecular basis for mutagenicity in meiosis may be related to the repair of meiotic DSBs by polymerases, or to the exposure of single-strand DNA to mutagenic agents during its repair.

The DNA2 nuclease/helicase is an estrogen-dependent gene mutated in breast and ovarian cancers.

Genomic instability, a hallmark of cancer, is commonly caused by failures in the DNA damage response. Here we conducted a bioinformatical screen to reveal DNA damage response genes that are upregulated by estrogen and highly mutated in breast and ovarian cancers. This screen identified 53 estrogen-dependent cancer genes, some of which are novel. Notably, the screen retrieved 9 DNA helicases as well as 5 nucleases. DNA2, which functions as both a helicase and a nuclease and plays a role in DNA repair and replication, was retrieved in the screen. Mutations in DNA2, found in estrogen-dependent cancers, are clustered in the helicase and nuclease domains, suggesting activity impairment. Indeed, we show that mutations found in ovarian cancers impair DNA2 activity. Depletion of DNA2 in cells reduces their tumorogenicity in mice. In human, high expression of DNA2 correlates with poor survival of estrogen receptor-positive patients but not of estrogen receptor-negative patients. We also demonstrate that depletion of DNA2 in cells reduces proliferation, while addition of estrogen restores proliferation. These findings suggest that cells responding to estrogen will proliferate despite impaired in DNA2 activity, potentially promoting genomic instability and triggering cancer development.

Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis.

Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30 We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed.

DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase.

Budding yeast cells suffering a single unrepaired double-strand break (DSB) trigger the Mec1 (ATR)-dependent DNA damage response that causes them to arrest before anaphase for 12-15 h. Here we find that hyperactivation of the cytoplasm-to-vacuole (CVT) autophagy pathway causes the permanent G2/M arrest of cells with a single DSB that is reflected in the nuclear exclusion of both Esp1 and Pds1. Transient relocalization of Pds1 is also seen in wild-type cells lacking vacuolar protease activity after induction of a DSB. Arrest persists even as the DNA damage-dependent phosphorylation of Rad53 diminishes. Permanent arrest can be overcome by blocking autophagy, by deleting the vacuolar protease Prb1, or by driving Esp1 into the nucleus with a SV40 nuclear localization signal. Autophagy in response to DNA damage can be induced in three different ways: by deleting the Golgi-associated retrograde protein complex (GARP), by adding rapamycin, or by overexpression of a dominant ATG13-8SA mutation.

The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage.

A single HO endonuclease-induced double-strand break (DSB) is sufficient to activate the DNA damage checkpoint and cause Saccharomyces cells to arrest at G(2)/M for 12-14 h, after which cells adapt to the presence of the DSB and resume cell cycle progression. The checkpoint signal leading to G(2)/M arrest was previously shown to be nuclear-limited. Cells lacking ATR-like Mec1 exhibit no DSB-induced cell cycle delay; however, cells lacking Mec1's downstream protein kinase targets, Rad53 or Chk1, still have substantial G(2)/M delay, as do cells lacking securin, Pds1. This delay is eliminated only in the triple mutant chk1Delta rad53Delta pds1Delta, suggesting that Rad53 and Chk1 control targets other than the stability of securin in enforcing checkpoint-mediated cell cycle arrest. The G(2)/M arrest in rad53Delta and chk1Delta revealed a unique cytoplasmic phenotype in which there are frequent dynein-dependent excursions of the nucleus through the bud neck, without entering anaphase. Such excursions are infrequent in wild-type arrested cells, but have been observed in cells defective in mitotic exit, including the semidominant cdc5-ad mutation. We suggest that Mec1-dependent checkpoint signaling through Rad53 and Chk1 includes the repression of nuclear movements that are normally associated with the execution of anaphase.

Inactivation of Ku-mediated end joining suppresses mec1Delta lethality by depleting the ribonucleotide reductase inhibitor Sml1 through a pathway controlled by Tel1 kinase and the Mre11 complex.

RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Delta, but not rad53Delta, lethality. We show that suppression of mec1Delta lethality is not due to Ku--associated telomeric defects but rather results from the inability of Ku- cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Delta lethality is also suppressed by lif1Delta, which like yku70Delta and yku80Delta, prevents nonhomologous end joining. The viability of yku70Delta mec1Delta and yku80Delta mec1Delta cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.

INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.

While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double-strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (gamma-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or gamma-H2AX results in reduced INO80 recruitment to the DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code.

DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain.

The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms. A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals. Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion. Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding. Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion. We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.

Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break.

BACKGROUND: In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form gamma-H2AX) and accumulate repair proteins at or near DSBs. To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between gamma-H2AX formation and repair protein recruitment remains to be defined. RESULTS: We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae. Break induction provoked rapid gamma-H2AX formation and equally rapid recruitment of the Mre11 repair protein. gamma-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most gamma-H2AX formation was dependent on Tel1 and Mre11. gamma-H2AX formed in a large (ca. 50 kb) region surrounding the DSB. Remarkably, very little gamma-H2AX could be detected in chromatin within 1-2 kb of the break. In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break. CONCLUSIONS: Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.

Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase.

In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Delta cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Delta is suppressed in mec1Delta strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Delta cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.