This application addresses the interactions between homologous chromosomes during meiosis in yeast. (I) DSB-independent pairing. We will use FISH to probe DSB-independent homolog pairing with respect to relative abundance in R- and G-bands, relationship to sister chromatid cohesion and involvement of chromatin structure proteins. We will use """"""""Capturing Chromosome Conformation"""""""" (3C) methodology to identify sites of pairing contacts. We will carry out a pilot experiment to investigate a possible new assay for pairing-defective mutants. (II) Initiation of recombination: the DSB transition. We will investigate whether pre-DSB recombinosomes are physically associated with their underlying chromosome axes by 3C methodology and genetic studies. We will also use 3C methodology to identify nascent DSB/partner interactions and to explore homolog/sister discrimination. We will further explore constraints governing the number of DSBs that can occur at a single locus in any give meiotic nucleus. (III) Later stages of recombination. We will further explore meiosis in mutants that affect the crossover control transition, with attention to important effects of temperature. We will continue analysis of the bouquet stage in wild type and selected mutants. We will continue analysis of the role of Mlh3 for meiotic recombination. We will examine the phenotypes of mutations suspected to affect conversion of single-end invasions to double Holliday junctions at mid-pachytene. And we will continue to investigate which topological isomer(s) of double Holliday junctions occur during meiosis. (IV) Meiotic chromosome structure and mechanics. We will further explore chromatin/axis/sister interplay revealed by our recent studies. We will use 3C methodology to investigate physical properties of mid-prophase chromosomes. We will examine the dynamics of synaptonemal complex twisting in vivo. We will begin to develop methods for isolating, analyzing and physically manipulating pachytene chromosomes in vitro.
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