Department of Biomedical Sciences
Contact Information: Email: firstname.lastname@example.org; Phone: 607-253-4301
Sponsor: NIH - National Institute of Child Health and Human Development (NICHD)
Grant Number: 3 R01 HD041012-09S1
Title: The MLH1-MLH3 Heterodimer in Mammalian Meiotic Recombination and Gametogenesis: Research Supplement to Promote Diversity
Annual Direct Cost: $15,824
Project Period: 02/02/2012-03/31/2014
DESCRIPTION (provided by applicant): The aim of the proposal is to understand how the MLH1-MLH3 heterodimer regulates the placement and integrity of crossovers in mammalian germ cells with the hypothesis that unique regulatory factors, both intrinsic and extrinsic to MLH1-MLH3, are in place to ensure the appropriate levels of crossing over. Aim 1 is focused on exploring the "intrinsic" regulation of MLH1-MLH3 function, by analysis of Mlh1-ATPase deleted mice, and by the generation and analysis of Mlh3-Endonuclease domain deleted mice. In Aim 2, we will explore the extrinsic regulation of MLH1-MLH3 by focusing on their associations with two major components of the recombinogenic machinery, Bloom syndrome mutated (BLM) protein and MUS81, both of which are known to interact functionally with each other and with the MMR pathway in many systems. Interactions between MLH1-MLH3 and BLM will be tested by analyzing mice harboring a conditional deletion at the Blm locus. BLM is RecQ helicase and is hypothesized to function in a restrictive role with respect to crossover function. In this respect, BLM and MLH1-MLH3 would act in opposition to modulate crossover frequency and distribution. The interaction between MLH1-MLH3 and MUS81 will be explored using Mus81-/- mice since this endonuclease has been shown, in yeast, to mediate an alternate crossing over pathway that is independent of MLH1-MLH3. The importance of this study is underscored by our recent observation that approximately 10% of crossovers in the mouse are MLH1-MLH3 independent, suggesting that the MUS81 pathway might functionally replace MLH1-MLH3-driven crossover mechanisms in this cohort. A second part of this aim will test interactions between EXO1, a downstream co-ordinator of MMR function, and MUS81/BLM. Finally, in aim 3, we will utilize a tandem affinity purification (TAP) system to identify protein complexes containing MLH1 and MLH3 by generating Mlh3-/- BAC transgenic mice containing a TAP-tagged Mlh3 fusion transgene. This will enable us to isolate meiotically-relevant native proteins that interact with MLH3 on meiotic chromosomes for subsequent functional analysis. Together, these studies will contribute substantially to our knowledge of meiosis and, more specifically, to the important role of MLH1-MLH3 therein.