Investigating Mammalian Meiosis: The Role Of Mismatch Repair Proteins And Their Interactors
Prophase I is the defining stage of meiosis when chromosomes must first pair with their homologous partner, then synapse, and undergo precisely controlled reciprocal recombination. Due to the complexity of the process, meiotic recombination requires highly ordered cooperation from various proteins, including the mismatch repair (MMR) protein family. Mouse MLH3 belongs to the MutL homolog family that functions as effector molecules for MMR. Research has suggested that MLH3 has critical roles in both DNA mismatch repair and meiosis. In the research for this thesis, I investigated two unique structural features of mouse MLH3: the potential endonuclease domain DQHA(X)2E(X)4E, and the large mammalian-specific region within exon 2. To investigate the function of the conserved endonuclease domain of MLH3, a transgenic mouse line containing a point mutation in this potential endonuclease domain was made. I hypothesized that, due to the conservation of this domain, disruption of this domain would lead to the abolishment of normal meiotic progression in vivo. To explore the function of the exon 2 region of mouse MLH3, I performed yeast two-hybrid assay and identified nine possible interacting partners of this region. To further screen for key sub-motifs within this region, a microsatellite instability reporter assay was tested. It is hypothesized that the unique region in exon 2 is important for the function of mouse MLH3 in maintaining genome integrity, and confers mammalian-specific functions to MLH3 in higher eukaryotes. Mouse CNTD1 is a newly identified cyclin-related protein. Its worm ortholog, COSA-1, functions in conjunction with the MMR pathway to process crossovers during meiosis. To explore the role of mouse CNTD1 in mammalian gametogenesis, I generated Cntd1 gene targeted mice. Consistent with the findings in C. elegans, deletion of Cntd1 in mice caused severe defects in meiotic CO formation, which leads to sterility in both males and females. No epididymal sperm were found in Cntd1 mutant males, and mutant females underwent severe oocyte-depletion after puberty. These data indicate a pivotal role for CNTD1 in regulating meiotic COs, possibly by helping select the sites of late recombination nodules through promoting or stabilizing other Class I CO-promoting proteins on meiotic chromosomes.
Mouse meiosis; mismatch repair protein; meiotic recombination; mlh3; cntd1
Weiss, Robert S.; Wolfner, Mariana Federica; Fortune, Joanne Elizabeth
Ph.D. of Physiology
Doctor of Philosophy
dissertation or thesis