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). Our previous studies have shown that the Sgs1 helicase, a homolog of the human BLM helicase, is a central regulator of meiotic recombination intermediate metabolism. In wild type cells, NCOs are formed by a process (synthesis-dependent strand annealing, SDSA) that does not involve stable recombination intermediates, while COs are formed by the resolution of stable, Holliday junction-containing recombination intermediates (called joint molecules or JMs), via a mechanism that requires the Mlh1-Mlh3 mismatch repair protein complex that is part of a meiosis-specific chromosome structure call the synaptonemal complex (SC). Normal meiotic JM resolution is independent of structure-selective nucleases (Mus81-Mms4, Yen1 and Slx1-Slx4), nucleases that resolve JMs during the mitotic cell cycle. In contrast, in sgs1 mutant cells, both COs and NCOs are formed by JM resolution that is structure-selective nuclease-dependent and Mlh1/Mlh3-independent. These findings point to a central role for Sgs1 in regulating the meiotic recombination. By disassembling branched recombination intermediates, Sgs1 drives events towards either early NCO formation by SDSA 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. Sgs1 is normally found in association with topoisomerase III and its partner protein, Rmi1. We examined the roles of topoisomerase III (Top3) and Rmi1 by making mutants that are specifically depleted for these proteins during meiosis. Meiotic phenotypes of meiosis-specific top3 and rmi1 mutants closely parallel those of sgs1 mutants, with a single exception. While JMs formed in sgs1 mutants are completely resolved by the end of meiosis, rmi1 and top3 mutants contained a fraction of JMs that remain unresolved. These findings suggest that Sgs1-meidated regulation of meiotic recombination occurs through the action of the trimeric Sgs1-Top3-Rmi1 complex, but that the Top3/Rmi1 heterodimer also has an independent role in resolving some of the JMs that form during meiosis. We are currently testing the hypothesis that this reflects the decatenating activity of Top3/Rmi1. Chromosome context determines meiotic recombination mechanism. Meiotic DSBs are formed by the Spo11 nuclease, and are formed and are repaired in the context of a meiosis-specific chromosome structure, the chromosome axis, 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, in a locus-specific manner. In a region of the genome that is axis-enriched, VDE-DSBs are repaired by mechanisms that closely resemble those for Spo11-catalyzed DSBs. In contrast, in a region that is axis-depleted, VDE-DSBs are repaired by mechansism that closely resemble those seen in mitotic cells. These results point to a critical role for chromosome structure in determining the pathways that will be used to repair DNA damage.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005268-26
Application #
8762988
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2013
Total Cost
$1,046,262
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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