Meiosis forms haploid gametes form diploid germ-line cells. During the first meiotic division, sister- chromatids remain connected while homologous chromosomes separate. For homologs to be accurately segregated, they must pair and become connected by at least one crossover. At least 12 proteins promote crossing-over during meiosis. Amongst these is Zip3, a conserved meiosis-specific protein that contains a RING-finger, which is a signature of ubiquitin and SUMO E3 ligases. Consistent with E3 ligase function, I have shown that the RING-finger is essential for Zip3 function and that Zip3 undergoes autocatalytic post- translational modification. I hypothesize that ZipS-promoted post-translational modification of recombination and chromosomal proteins promotes meiotic crossing-over. The long-term goal of this project is to understand how Zip3 promotes meiotic crossing-over.
The Specific Aims are: 1. To demonstrate that Zip3 has ubiquitin or SUMO E3 ligase activity in vitro an in vivo. Mouse and yeast Zip3 proteins will be purified and their ability to promote SUMO or ubiquitin conjugation will be tested in vitro using purified components. Preliminary studies indicate that S. cerevisiae Zip3 undergoes self-catalyzed post-translational modification in vivo. The nature of this modification will be determined using molecular and genetic approaches. 2.
Specific Aim 2 : To analyze the function ofZipS automodification. To prevent Zip3 modification, modified lysine residues will be changed to arginine using site-directed mutagenesis. Meiotic recombination and homolog pairing will then be monitored in these mutant strains using genetic analysis, specialized DNA physical assays and immunofluorescence. 3. To identify in vivo substrates of yeast Zip3. A series of candidate proteins will be examined for the presence of ZipS-dependent post-translational modification. Relevance: Chromosome pairing and recombination are fundamental to sexual reproduction and chromosome repair. Defects in recombination have been linked to human infertility, miscarriage and genetic diseases, particularly cancer. An understanding of the mechanism and regulation of recombination will therefore help us better understand the etiology of these diseases. ? ? ?
