Meiotic double-strand DNA break (DSB) repair by homologous recombination occurs via multiple processes defined by distinct decisions points. One important decision involves partner choice, between recombining with the sister chromatid (the dominant repair partner during mitosis) or with the homolog (the homologous chromosome of different parental origin, the preferred partner during meiosis). Another important decision involves recombination pathway choice, between producing crossovers, where flanking chromosome sequences are exchanged, or noncrossovers. A signature contribution of our group was the demonstration that crossover and noncrossover recombination proceed via different mechanisms that diverge after initial stages of strand invasion, and that feature different biochemical activities and genetic requirements. Work during previous review periods had shown that the conserved Sgs1-Top3-Rmi1 helicase-topoisomerase complex (STR) is responsible for partitioning early recombination events between noncrossover and crossover pathways. Sgs1-Top3-Rmi1 is the yeast homolog of the mammalian BLM helicase-Top3alpha-BLAP75 complex, implicated in cancer avoidance and recombination control in humans. We showed that all three members of the yeast complex are essential for normal recombination partner choice and for population of regulated meiotic crossover and noncrossover recombination pathways. Based on these findings, we hypothesized that STR, by promoting frequent disassembly of early strand invasion intermediates, acts as a chaperone for early recombination intermediates. We hypothesized that these repeated cycles of strand invasion and disassembly would result in template switching, which in turn would lead to recombinants with mosaic parental strand contributions. This hypothesis has now been confirmed by high-throughput sequencing of recombinants that occur in a highly polymorphic test interval; more than 2/3 of recombinants display clear evidence for template switching, multiple strand invasions, or both. In addition, we uncovered evidence for activities specific to the crossover pathway, including branch migration (2/3 of crossovers) and exonucleolytic gap-formation (1/3 of crossovers). Current work is aimed at determining the proteins responsible for these activities. Other work is aimed at confirming branch migration by mapping the location of Holliday junctions in recombination using a novel method we have developed to specifically purify Holliday junction-containing intermediates. In addition, prompted by observations that mutants in the SUMO-ligase activity of the conserved Smc5-Smc6 DNA repair complex show meiotic recombination defects similar to STR complex mutants, we are testing the possibility that Smc5-Smc6-mediated SUMOylation of STR proteins is important for their meiotic activity. Studies of meiotic recombination in mutants lacking STR components also identified an Sgs1-independent role for Top3-Rmi1, in resolving strand entanglements that accumulate in recombination intermediates. Using a procedure call return-to-growth, where recombination intermediates are accumulated during wild-type meiosis and then resolved in a subsequent mitotic cell cycle, we have shown that Top3-Rmi1, but not Sgs1, is not required to fully resolve recombination intermediates. Unresolved recombination intermediates that remain in the absence of Top3-Rmi1 induce a DNA damage checkpoint that arrests cell cycle progression; we currently are determining how these intermediates are recognized and how they induce the DNA damage response. Finally, we are studying how chromosome structure, specifically the meiotic chromosome axis, contributes to the regulation of recombination. A meiosis-specific subset of chromosome axis components, the Hop1 and Red1 proteins, are important for meiotic DSB formation and partner choice, and are enriched in some regions of the genome relative to others. Using a novel method to recruit axis proteins to regions that are normally depleted of these proteins, we have shown that high concentrations of the Hop1 protein are necessary and sufficient for meiotic DSB formation. We are currently determining the mechanism by which Hop1 promotes DSB formation, and if it impacts subsequent steps of meiotic recombination.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Kaur, Hardeep; Ahuja, Jasvinder S; Lichten, Michael (2018) Methods for Controlled Protein Depletion to Study Protein Function during Meiosis. Methods Enzymol 601:331-357
Lichten, Michael (2017) Proteasomes on the chromosome. Cell Res 27:602-603
Vaid, Rajni; Dev, Kamal; Lichten, Michael et al. (2016) Generation of an inducible system to express polo-like kinase, Cdc5 as TAP fusion protein during meiosis in Saccharomyces cerevisiae. 3 Biotech 6:185
Medhi, Darpan; Goldman, Alastair Sh; Lichten, Michael (2016) Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis. Elife 5:
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
Lichten, Michael (2015) Molecular biology. Putting the breaks on meiosis. Science 350:913
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 57:583-594
Borde, Valérie; Lichten, Michael (2014) A timeless but timely connection between replication and recombination. Cell 158:697-8
De Muyt, Arnaud; Jessop, Lea; Kolar, Elizabeth et al. (2012) BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism. Mol Cell 46:43-53
Lichten, Michael; de Massy, Bernard (2011) The impressionistic landscape of meiotic recombination. Cell 147:267-70

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