Progress has been made 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. 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. Because further mechanistic insight would be gained by knowing precise DSB locations, we are developing high-throughput sequencing methods to determine the genome-wide location of meiotic DSBs at single-nucleotide resolution. Our initial aim is to apply this approach to the study of meiotic DSBs, but also to obtain high-resolution, genome-wide maps of spontaneous DNA damage in growing cells, and DNA damage induced by carcinogenic and cancer chemotherapeutic agents.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 protein structures that are part of the synaptonemal complex (SC). SC breakdown and JM resolution as COs are triggered by expression of the Cdc5 polo-like kinase upon 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 nuclease for formation. Two other nucleases, Yen1 and Slx1-Slx4, were identified as potential JM resolvases, but their roles in meiotic recombination were not determined. We found 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. 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. Prompted by our 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, both NCOs and COs are Cdc5-dependent, and Mus81-Mms4, Yen1 and Slx1-Slx4 are redundantly required to form both NCOs and COs. These findings point to a central role for Sgs1 in regulating the meiotic recombination. By disassembling branched recombination intermediates, Sgs1 drives these events towards either early NCO formation or towards JM and subsequent CO formation in the context of the SC, which protects JMs from Sgs1. In the absence of Sgs1, the vast majority of events form JMs, which are then resolved in an apparently unbiased manner,producing both NCOs and COs. We are currently examining the roles of topoisomerase III, which is found in complex with Sgs1, in regulating recombination intermediate metabolism.Partner choice during meiotic recombination. Recombination can occur between sister chromatids or between homologous chromosomes of differing parental origin, called homologs. Inter-sister recombination, is the predominant form of recombination during the mitotic cell cycle, while interhomolog recombination, which is required to pair homologs and to ensure their disjunction at the first meiotic division, predominates during meiosis. We have shown that the increased level of interhomolog recombination during meiosis results from a modest (3-fold) reduction in levels of intersister recombination. Current research is aimed at characterizing features of meiotic chromosome structure that enforce the switch in partner choice that occurs between mitosis and meiosis.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. Two mechanisms could contribute to these differences. First, the presence of multiple DSBs and of meiosis-specific recombination proteins might change recombination biochemistry. Second, meiotic DSBs form in and are repaired in the context of meiotic chromosome structures, which could influence recombination outcome. To test this, we are studying repair of DSBs catalyzed by a meiosis-specific endonuclease VDE, which can form DSBs outside the meiotic structural context. We found VDE-DSB repair significantly differs from that of normal meiotic DSBs. VDE-DSBs show a greater fraction of NCOs than do normal meiotic DSBs, and the COs that form are largely independent of the Cdc5 kinase, which is required for normal meiotic CO formation. These differences in repair of VDE-DSBs and normal meiotic DSBs suggests that the meiotic chromosome structure is central to both partner choice and DSB repair mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005268-25
Application #
8552569
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2012
Total Cost
$1,143,603
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Xue, Xiaoyu; Papusha, Alma; Choi, Koyi et al. (2016) Differential regulation of the anti-crossover and replication fork regression activities of Mph1 by Mte1. Genes Dev 30:687-99
Kaur, Hardeep; De Muyt, Arnaud; Lichten, Michael (2015) Top3-Rmi1 DNA single-strand decatenase is integral to the formation and resolution of meiotic recombination intermediates. Mol Cell 57:583-94
Lichten, Michael (2015) Molecular biology. Putting the breaks on meiosis. Science 350:913
Borde, Valérie; Lichten, Michael (2014) A timeless but timely connection between replication and recombination. Cell 158:697-8
Dayani, Yaron; Simchen, Giora; Lichten, Michael (2011) Meiotic recombination intermediates are resolved with minimal crossover formation during return-to-growth, an analogue of the mitotic cell cycle. PLoS Genet 7:e1002083
Sourirajan, Anuradha; Lichten, Michael (2008) Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis. Genes Dev 22:2627-32
Lichten, Michael (2008) Genomics: Thoroughly modern meiosis. Nature 454:421-2
Jessop, Lea; Lichten, Michael (2008) Mus81/Mms4 endonuclease and Sgs1 helicase collaborate to ensure proper recombination intermediate metabolism during meiosis. Mol Cell 31:313-23