During the past year, we have made progress in the following areas: Genome-wide mapping of DNA damage. Meiotic recombination is initiated by DNA double-strand breaks (DSBs), whose location and time of formation are tightly controlled. Our previous work developed a novel method to isolate intermediates in DSB repair, and applied this method to a genome-wide map of meiotic DSBs, based on microarray analysis, with a resolution of about 500 nucleotides. While this method had sufficient resolution to document overall DSB distributions, further mechanistic insight would be gained by knowing precise DSB locations. We are developing a method to determine the genome-wide location of meiotic DSBs at single-nucleotide resolution, using a modified high-throughput sequencing protocol. Our initial aim is to determine the substrate requirements of proteins that form DSBs, the factors that control DSB location, frequency, and timing, and how these factors change over evolutionary time. Future work will modify this approach to allow high-resolution, genome-wide maps of spontaneous DNA damage in growing cells, and DNA damage induced by carcinogenic and cancer chemotherapeutic agents. Partner choice during meiotic recombination. Recombination can occur between sister chromatids or between homologous chromosomes of differing parental origin, called homologs. Inter-sister recombination, which is the predominant form of recombination during the mitotic cell cycle, was thought to be greatly reduced during meiosis, since interhomolog recombination is required to pair homologous chromosomes and to ensure their disjunction at the first meiotic division. We have now show that inter-sister recombination occurs much more frequently that previously thought during meiosis, and that the increased level of interhomolog recombination that occurs during meiosis results from a modest (3-fold) reduction in the rate of intersister recombination. These findings are prompting a revision of the nature of recombination partner choice control, in particular the regulatory mechanisms that limit intersister and promote interhomolog recombination. Recombination intermediate metabolism during meiosis and the mitotic cell cycle. Recombination produces both crossovers (COs) and noncrossovers (NCOs). Previous studies have shown that, in wild-type budding yeast meiosis, most COs are produced by the resolution of bi-parental recombination intermediates (joint molecules, JMs) that associate with and require the integrity of specialized protein assemblages that are part of the synaptonemal complex (SC). SC breakdown and JM resolution as COs are both triggered by the Cdc5 polo-like kinase, which is expressed as cells exit from the pachytene stage of meiosis. In contrast, most NCOs are SC- and Cdc5-independent, and are formed well before exit from pachytene. A few COs are also SC-independent, and require the structure-selective Mus81-Mms4 endonuclease for formation. Two other yeast nucleases, Yen1 and Slx1-Slx4, have been identified as potential JM resolvases, but their contributions to meiotic recombination were not determined. We find that, in wild-type cells, Mus81-Mms4 and Yen1 act redundantly to resolve a minor fraction (10-20%) of JMs and to produce a similarly minor fraction of COs, with little, if any, contribution from Slx1-Slx4. Therefore, a yet-unidentified, Cdc5-activated nuclease resolves the majority of meiotic JMs and forms the majority of COs in wild-type cells. Sgs1, the budding yeast BLM helicase homolog, has been identified as regulating aspects of recombination, both in meiosis and in the mitotic cell cycle. sgs1 mutation partially suppresses the JM- and CO-deficiency of SC-defective mutants, and renders the majority of COs dependent upon Mus81-Mms4. Prompted by recent findings suggesting that Sgs1 disassembles JMs during the mitotic cell cycle, we re-examined meiotic recombination in sgs1 mutant cells. While NCOs are Cdc5-independent and appear before COs in wild-type cells, NCOs and COs appear at the same time in sgs1 mutants. In sgs1 mutants, Cdc5 expression triggers both NCO and CO formation, and Mus81-Mms4 and Yen1 are required to form the majority of NCOs as well as the majority of COs. These findings point to a central role for Sgs1 in regulating the meiotic recombination events that are not directed towards SC-dependent JM- and CO-formation. Sgs1 drives these events towards early NCO formation;in the absence of Sgs1, the vast majority of these events form JMs, which are then resolved in an apparently unbiased manner (producing both NCOs and COs) when cells exit from pachytene. These findings also suggest that all meiotic endonuclease-type JM resolvases, including Mus81-Mms4 and Yen1, are activated by the Cdc5 polo-like kinase. We have also examined the role of Sgs1 and structure-selective nucleases in JM metabolism in mitotic cells, using the ability of yeast cells to exit meiosis and return to the mitotic cell cycle upon nutrient restoration. This procedure, called return to growth (RTG), allows us to accumulate recombination intermediates during meiosis, return to the mitotic cell cycle, and examine their resolution under mitotic conditions. We find that the majority of JMs are resolved as NCOs, not COs, during the first 2-2.5 hr after RTG, in a manner that requires Sgs1 helicase but not Mus81-Mms4, consistent with this JM resolution occurring via a helicase-dependent mechanism called dissolution. After 2-2.5 hr, coincident with Cdc5 expression, JMs are resolved as both COs and NCOs in a manner that is largely dependent upon Mus81-Mms4. This findings point to remarkable mechanistic parallelsbetween meiotic and mitotic recombination. In both, early intermediates are channeled towards NCO recombination by Sgs1 helicase activity;in both, resolution of JMs that escape Sgs1 activity are resolved by Cdc5-activated nucleases. The two processes differ in that meiotic cells use synaptonemal complex components to protect a class of JMs from Sgs1 activity, and to direct their resolution predominantly towards CO formation. Contribution of chromosome structure to the outcome of meiotic recombination Mitotic recombination is initiated by spontaneous lesions, occurs mainly with sister chromatids, and infrequently produces COs. Conversely, meiotic recombination is initiated by programmed DSBs, occurs mainly with the homolog, and frequently produces COs, which are needed for proper homolog segregation during meiosis I. These features of meiotic recombination could derive from two influences. First, the presence of multiple DSBs and of meiosis-specific recombination proteins may cause a global shift in recombination biochemistry;second, meiotic DSBs form in and are repaired in the vicinty of meiosis-specific chromosome structures, which may influence recombination outcome. To test this, we are studying repair of DSBs catalyzed by a meiosis-specific endonuclease VDE, which has the potential to form DSBs outside the meiotic structural context. We found VDE-DSB repair significantly differs from that of normal meiotic DSBs. VDE-DSBs displayed a reduced likelihood of IH repair and show a greater fraction of NCOs than do normal meiotic DSBs. Moreover, VDE-initiated NCOs and COs both form in cells that do not express Cdc5, suggesting that VDE-initiated recombination intermediates are resolved by mechanisms that differ from those used in normal meiotic recombination. We propose that repair of VDE-DSBs and normal meiotic DSBs are different because normal meiotic recombination occurs in the context of the chromosome axis, while VDE DSBs are repaired off the axis. This suggests that the role of the meiotic chromosome axis is central to both partner choice and DSB repair mechanisms.
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