Epic Search for the Perfect Partner: MutSgamma Promotes Robust Meiotic Recombination and Homolog Pairing in Mouse Spermatocytes

Melissa Noelle Frasca, BS (Advisor: Francesca Cole Bergemann, PhD)

For diploid organisms, like mice and humans, to sexually reproduce, their cells first go through a reductional division called meiosis. Meiosis creates haploid gametes like sperm and eggs from diploid parental cells to ensure that upon sexual reproduction the genome doesn’t double with each generation. For parental chromosomes (homologs) to accurately segregate in meiosis I they must be physically connected to each other to provide tension across the metaphase plate. Homologs are physically attached together by a product of DNA repair by homologous recombination called crossovers, which involve a reciprocal exchange of homolog arms. However, before a crossover can be made, the homologs first have to come together and align along their lengths. This is a process called pairing. In humans and mice, among other organisms, meiotic recombination promotes but also relies upon pairing between homologs. Interhomolog pairing is required for completion of meiotic recombination. The mutual dependence and differential reliance between recombination and pairing in well-studied organisms has been difficult to deconstruct in the mammalian context.  For example, when trying to analyze pairing in organisms by cytology, synapsis is used as a poor proxy for pairing. Synapsis occurs when homologs are brought in closer juxtaposition and connected by a proteinaceous structure called the synaptonemal complex. Synapsis is achieved much later in meiotic prophase than pairing, but is also necessary for efficient interhomolog recombination in budding yeast and mammals confounding the relationship between recombination and pairing. The field has lacked the technology to directly measure pairing genome-wide and a previous graduate student in our lab was able to distinguish pairing from synapsis in wild type meiosis. Overall, pairing, synapsis, and recombination are interconnected and mutually dependent upon each other. The goal of my thesis project was to study the relationship between recombination and pairing and in particular the role of MutSgamma in both processes.

In budding yeast, MutSgamma, a heterodimer between MSH4 and MSH5 promotes crossover-specific recombination by protecting precursors, and in many organisms plays roles in pairing and synaptonemal complex formation. In mouse, it has been shown that MutSgamma is required for crossover formation and these mutants also show defects in pairing and synapsis (shown by incomplete synapsis and/or synapsis between more than two chromosomes). As such, spermatocytes from these mutants apoptose and these mice are infertile. It has also been shown, both in budding yeast and in mouse, that mutations to the ATPase domain of MSH5 have similar defects. These mutants do not form crossovers and have similar pairing and synapsis defects, but a key difference is that this mutant can still form the complex between MSH4 and MSH5. MutSgamma has been shown in vitro to specifically bind to Holliday junctions, an intermediate that is often repaired as a crossover. It is thought that MutSgamma forms a sliding clamp to encircle crossover-specific intermediates in order to stabilize and protect these intermediates from dissolution.           

As mentioned, interhomolog recombination and pairing are mutually dependent upon each other. However, which steps in recombination are required for pairing and how much interhomolog pairing is necessary for proficient recombination is not well understood, especially in mammals. My thesis focuses on understanding some of the requirements for accurate interhomolog recombination and pairing during meiotic prophase I. I use recombination and cytological assays to infer the role of MutSgamma in mouse spermatocytes. I find in two alleles of Msh5 – a null and one bearing a mutation in its ATPase domain - that spermatocytes are severely compromised for recombination producing only a small fraction of noncrossovers. However, they are more proficient in interhomolog pairing particularly on the longer chromosomes than spermatocytes lacking meiotic recombination entirely. I propose that MutSgamma plays an earlier role in mouse than in budding yeast to stabilize D-loops upstream of all interhomolog recombination. Further, that nascent recombination interactions can promote successful interhomolog pairing despite not completing recombination.

Advisory Committee:

• Francesca Cole Bergemann, PhD, Chair
• Swathi Arur, PhD
• Georgios Karras, PhD
• Angela Ting, PhD
• Richard Wood, PhD