MutS and MutL proteins in mammalian meiotic recombination and DNA repair

Abstract

In addition to their role in somatic DNA repair mammalian MutS and MutL mismatch repair proteins play critical meiosis-specific roles facilitating the formation of reciprocal exchanges between homologous chromosomes (crossovers). MutS homologs 4 and 5 (MSH4 and MSH5) localize to nascent crossover sites in zygonema, followed by MutL homologs 1 and 3 (MLH1 and MLH3) that form MutLgamma heterodimer and localize to paired homologous chromosomes in pachynema marking the final crossover sites, while a complex of MLH 1 and PMS2 plays major mismatch repair role. We hypothesized the existence in mammalian germ cells of MutLgamma-dependent major crossover pathway that is responsible for the formation of most crossovers; the MLH1-independent mode of targeting for MLH3 protein and that PMS2 protein is dispensable for crossover formation but may play other meiosis-specific repair role. Immuno-cytological analysis in wild type and MutL homologs knockout mice (Mlh1-, Mlh3- and Pms2-null) confirmed quantitatively the crossover sites-specific localization of MutLgamma, MLH1-independent targeting of MLH3 to nascent crossover sites and unperturbed crossing over and MutLgamma-localization in absence of PMS2. We discovered novel association of MutL homologs and the repair-specific MutSbeta complex (MSH2-MSH3) with DNA repeat regions in mouse spermatocytes, including the centromere satellite DNA and Y-chromosome, confirmed by chromatin immunoprecipitation (ChIP) analysis. We next characterized the effect on crossover process of the ablation of MLH3 by PCR analysis of the recombination frequency and crossover product mapping at the mouse Psmb9 hotspot of meiotic recombination. MLH3 ablation resulted in 10-20 fold reduction of crossover frequency at the hotspot with high incidence of unusual complex structures among residual crossovers. Finally, we tested the physical association of the MutL crossover complex with recombination site DNA in isolated mouse pachytene spermatocytes by ChIP-chip method. Major component of the complex, MLH3, specifically associated with genomic DNA at the Psmb9 recombination hotspot in pachynema. The association boundaries coincided with the hotspot boundaries previously found genetically, while no association of MLH3 with adjacent recombination-cold DNA was found. In summary, this thesis characterizes and specifies the functions of mismatch repair proteins in meiotic crossing over and DNA repair and opens new avenues in investigation of mammalian meiosis.