Neil Hunter uses yeast and mice to study homologous recombination, an essential chromosome repair process.
Hunting for Partners of the E3 Ligase, RNF212
Reciprocal exchange or crossing-over between parental chromosomes is essential for their accurate segregation at the first division of meiosis. Defects in crossing-over contribute to infertility, aneuploid diseases such as Down syndrome, and the maternal-age effect (decreased fertility and increased risk of birth defects in older women). Variants of the human RNF212 gene have been linked to variation in the rate of crossing-over. We have shown that mouse Rnf212 is essential for crossovers and shows an unusual haploinsufficient phenotype of reduced crossing-over in heterozygous animals. Preliminary data indicate that RNF212 is an E3-ligase for the small protein modifier, SUMO. This project aims to identify and characterize proteins that interact with RNF212, a putative E3 ligase for small protein modifiers such as ubiquitin or SUMO. Yeast two-hybrid, immunoprecipitation, and biochemical approaches will be used to test candidate interacting proteins. There will also be opportunities to learn immunofluorescence and super-high-resolution cytology techniques.
Roles of Proteasomal Protein Turnover in Regulating Meiotic Recombination
Homologous recombination plays an essential role in the pairing and segregation of paternal and maternal chromosomes during meiosis. Meiotic recombination initiates with the programmed formation of hundreds of DNA double-strand breaks. A few breaks are assigned a crossover rate in such a way that each pair of chromosomes obtains at least one crossover, as required for accurate segregation. We have obtained evidence that this crossover control process involves the differential stabilization of key recombination factors, which in turn stabilize DNA recombination intermediates. A role for ubiquitin-dependent protein turnover is invoked by these data. This project will address the role of the proteasome in the turnover of specific recombination factors. We will also take an unbiased approach to identify meiotic proteins that undergo ubiquitin-dependent turnover during meiotic prophase. The project will utilize genetic, cytological, biochemical, and mass spectrometry approaches.
Holliday Junction Resolution During Homologous Recombination
Homologous recombination is an essential chromosome repair process that maintains genome stability and facilitates chromosome segregation during meiosis. At the final step of homologous recombination, Holliday junction containing joint molecule DNA intermediates must be resolved to allow chromosomes to segregate. Joint molecule resolution is also highly regulated, especially during meiosis where crossovers must be formed to facilitate the accurate segregation of parental chromosomes. This project will explore the contributions of five resolving factors that we have recently shown to be involved in joint molecule resolution. The project will utilize molecular genetic approaches in budding yeast to understand the contributions of the different resolving factors as well as to explore their mechanism of action and their regulation. Techniques include yeast genetics, molecular biology, and electron microscopy.