Dr. Kleckner proposes to continue her analysis of meiotic chromosome structure and meiotic recombination in the yeast Saccharomyces cerevisiae. There are six specific aims: First, she plans to continue development of psoralen derivatives that will allow detection of unstable DNA-DNA interactions in mitosis and meiosis; genetic systems for the detection of such interactions will also be developed. Second, she will examine various aspects of double-strand break (DSB) formation; evidence indicates that the DSB is the initiating lesion for meiotic recombination. She will characterize a system in which the DSBs are formed in vitro. Mutants defective for in vivo formation of DSBs will be examined in the in vitro system, and she will attempt in vitro complementation between extracts made from different mutants. If such complementation experiments are successful, she will try to purify the complementing gene products. In preliminary experiments, she has evidence for an end-binding protein; she will attempt to purify this protein. She will determine whether the Rad50p localizes to nuclease hyper-sensitive sites in chromatin, and will look for mutations that suppress the rad50S mutant defect. Third, she will study steps of meiotic recombination that occur after formation of the DSB. In a previous study, she detected a recombination intermediate that had the properties expected for a double Holliday junction; she refers to these structures as joint molecules. She plans a detailed study of the morphology of the Holliday junction by electron microscopy. She will also determine whether sequences within the joint molecules have undergone mismatch repair. Joint molecules are formed between both sister chromatids and between homologs. She will examine both types of joint molecules in various mutant strains. She has identified a gene, SAS3, that appears to encode an RNA species required for progression through meiosis. She will determine whether this RNA localizes to meiotic chromosomes, and will identify proteins that interact with this RNA species. In most eukaryotes, crossovers suppress adjacent crossovers. The fourth specific aim is to study this phenomenon, interference, in detail. A system for analyzing interference genetically and physically will be developed, and she will isolate mutants that have elevated levels of interference. Dr. Kleckner will attempt to obtain zip1 mutants with normal recombination and interference, but defective synaptonemal complexes. The fifth specific aim is to identify and clone the DNA sequences located at the axis of the meiotic chromosomes. She will determine whether these sequences correlate with other features of interest in the meiotic chromosomes (for example, nuclease hyper-sensitive sites). The sixth and last aim is to determine whether the kinase domain of the Mek1p allows bypass of the arrest observed in dmc1/rad51/zip1 strains; such a bypass is observed in mek1 null mutant strains. She may also determine whether Mek1p colocalizes with Dmc1p and Rad51p.
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