Genetic recombination is essential for the accurate segregation of homologous chromosomes from one another in meiosis. Much of the meiotic recombination pathway is now understood at the molecular level, largely due to studies in S. cerevisiae. The final events in the pathway, however, remain very poorly understood, and few proteins have been identified as candidates for carrying out these events. The Drosophila mei-9 gene acts during these late stages. The MEI-9 protein is a component of a nucleotide excision repair (NER) endonuclease, which cuts DNA structures that undergo single-stranded to double-stranded transitions with a specific polarity. The human homolog of MEI-9 is XPF, a gene implicated in the hereditary disorder xeroderma pigmentosum, which is associate with skin cancer. An attractive model for the meiotic function of MEI-9 is that it cuts Holliday junctions to resolve recombination intermediates. Experiments in this proposal seek to test this model. Half-tetrads (two chromatids) of meiotic recombination events will be characterized molecular from both wild type and mei-9 mutants, providing information about the structure of the recombination intermediate and the manner in which it is resolved. MEI- 9 protein will be immunolocalized with anti-MEI-9 antibodies or with epitope tags. Fifteen mutant alleles of mei-9 will be characterized molecularly and genetically to learn about how the structure of the protein correlates with its function. The mus210 gene encodes the second subunit in Drosophila. Mutations in this gene disrupt NER, but not meiotic recombination. Hence, either these are separation-of-function alleles, or the activity of this protein is not required in meiosis. If the latter is true, this is the first case of separation of function between the two subunits. To test this, new mus210 mutations will be recovered and characterized. If MEI-9 does not require MUS210, it may be that MUS210 is present and dimerizes, but its activity is not required, or that the protein is present but does not dimerize with MEI-9, or that MUS210 is not present in meiotic cells. The expression of the protein will therefore be analyzed. In addition, expression of the protein will be forced, to determine whether it interferes with the MEI-9 function. Finally, potential meiosis-specific partners of MEI-9 will be identified.
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