Neil Hunter uses yeast and mice to study homologous recombination, an essential chromosome repair process.
Hunting for Partners of the E3 Ligases RNF212 and HEI10
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 genes RNF212 and HEI10 (a.k.a. CCNBIP1) have been linked to variation in the rate of crossing-over. We have shown that mouse Rnf212 and Hei10 are essential for crossovers and show an unusual dosage-sensitive phenotype of reduced crossing-over in heterozygous animals. Biochemical data indicate that RNF212 is an E3 ligase for the small protein modifier SUMO, whereas HEI10 is a ubiquitin ligase. Moreover, these proteins appear to play antagonistic roles with respect to the stability of recombination factors along synapsed chromosomes. This project aims to establish and characterize proteins that interact with RNF212 and HEI10. A number of candidate partners and substrates have been identified from unbiased and biased approaches, including yeast two-hybrid screenng and immunoprecipitation. Partner proteins will be confirmed and their functions analyzed using a vareity of cytological, molecular, genetic and biochemical approaches biology.
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 highly regulated, especially during meiosis, where crossovers must be formed to facilitate the accurate segregation of parental chromosomes. This project will analyze five resolving factors that we have shown account for essentially all Joint Molecule resolution in vivo. The project will utilize molecular genetic approaches in budding yeast to understand the mechanism and regulation of Joint Molecule resolution during meiosis. Techniques include yeast genetics, molecular biology, mass spectrometry, biochemsitry, and electron microscopy.
Holliday Junction Processing by MutS and MutL Proteins
During meiosis, hundreds of DNA double-strand breaks are induced and subsequently repaired by homologous recombination to facilitate the pairing and synapsis of homologous chromosomes. About 10% of repair events are associated with an exchange of chromosome arms called a crossover. In collaboration with cohesion between sister chromatids, crossovers connect homologous chromsome pairs, allowing their stable biorientation on the spindle and accurate segregation at the first meiotic division. Defective crossing over can result in sterility, aneuploidy, pregancy miscarriage, and birth defects such as Down syndrome. Crossovers form via DNA intermediates called double Holliday Junctions. These structures are resolved by nicking DNA strands with a defined orientation. The enzymatic machinery responsible for double Holliday Junction resolution was unknown until our recent analysis implicated several factors related to DNA mismatch repair: MutS complex MSH4-MSH5, MutL complex MLH1-MLH3, and the exonuclease EXO1. This project will investigate the biochemical properties of these factors in the recognition and processing of double Holliday Junctions. Techniques include protein expression and purification, a variety of electrophoretic and biophysical assays, and single-molecule biochemsitry.
Oocyte Quality Control and the Role of RNF212
Meiosis shows profound sexual dimorphism. In males, meiosis initiates during puberty and continues unabated throughout life, producing an essentially unlimited gamete pool. Defects in meiosis induce a robust arrest and apoptosis response. In females, only one round of meiosis occurs during fetal development and, as such, the oocyte number is finite. Three quality control processes determine the size of the oocyte pool at puberty: (1) fetal attrition, which is thought to cull oocytes that have experienced excessive LINE-1 transposon activity; (2) postpartum atresia, which culls oocytes that experienced problems with chromosome pairing and recombination; and (3) ongoing apoptosis of oocytes that experience damage or other cellular stress. In mouse, mutations in genes required for chromosome pairing and recombination, such as Spo11 and Msh4, invoke robust postpartum atresia resulting in the complete loss of the oocyte pool. We recently identified a key factor in this response, a SUMO E3-ligase called RNF212. In Rnf212 Spo11 and Rnf212 Msh4 double mutants, the oocyte pools are rescued, identifying RNF212 as a checkpoint factor for oocyte quality control. This project aims to understand how RNF212 functions in the checkpoint pathways that monitor meiotic processes and signal to the apoptotic machinery. Techniques include immunohistochemistry, imunocytology, mouse genetics, and molecular biology.