Homologous recombination is required during meiosis to promote accurate segregation of homologous chromosomes at the first meiotic division. Several proteins have been identified that re required for MEIOTIC RECOMBINATION in the YEAST Saccharomyces, but the specific role of these proteins in recombination is largely unknown. The long term goals of this work are to determine the molecular mechanism of meiotic recombination and how the cell cycle machinery coordinates recombination with other meiotic events. This project focuses on one meiotic protein, Dmcl, that is required for meiotic recombination. Dmc1 and its relative Rad51 share structural and functional homology to RecA protein. RecA is the central protein required fo recombinogenic REPAIR OF DNA DAMAGE in E. Coli. Yeast Rad51, and probably Dmc1 as well, share RecAs ability to promote strand exchange between two molecules. Dmcl protein will be purified and characterized to determine what activities it shares with RecA. The formation of recombination products requires numerous enzymatic activities in addition to those displayed by RecA. Many of the proteins required for meiotic recombination form a multi protein complex. Affinity chromatography and tandem mass spectroscopy will be used to identify proteins that form complexes with Dmcl during recombination. Time-course and mutational analyses will be used to characterize overall progress in the assembly and disassembly of functional recombination complexes. In addition, confocal microscopy will be used to examine individual recombination events in living cells using green fluorescent protein. The functions of Rad51 and Dmc1 are only partially redundant. We propose mutant screens designed to separate the redundant function of Dmc1 from its unique functions. These mutations will then be used to characterize the mechanistic features of meiotic recombination that distinguish it from mitotic recombinational repair. The function of Dmc1 is monitored by a checkpoint control mechanism that ensures recombination is complete before chromosome segregation occurs. We recently showed the genes required for checkpoint control have additional functions in synapsis and suppression of ectopic recombination. The relationship between these newly identified functions and checkpoint control will be examined.
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