Investigation of the Binding Interaction of S. cerevisiae MutS Homologs MSH2-MSH6 and MSH4-MSH5 with Holliday Junctions

Abstract

The MutS homolog family in eukaryotes (MSH2, MSH3, MSH4, MSH5 and MSH6) forms three heterodimeric protein complexes: MSH2-MSH3, MSH2-MSH6 and MSH4-MSH5. The heterodimer, MSH2-MSH6, plays fundamental roles in post replication mismatch repair and specifically recognizes mismatched base pairs and small insertion/deletion loops. Previous studies have indicated that the MSH4-MSH5 heterodimer participates in meiotic crossover and specifically binds to a homologous genetic recombination intermediate, the four-way junction (4WJ) or Holliday junction. The repair protein, MSH2-MSH6 also binds with high affinity to the 4WJ. In this work, we explore the different interactions and complexes formed between these two proteins and DNA 4WJs to potentially shed light on the different roles they play in Holliday junction processing. The protein-DNA interaction is investigated using gel mobility shift assay and fluorescence anisotropy methods to determine binding affinity. Our work supports the previous finding that MSH2-MSH6 binds to the 4WJ with comparable affinity to mismatched DNA. Fluorescence measurements are being used to determine the stoichiometry of protein binding to the junction. Resonance energy transfer measurements are being used to determine the conformational changes induced upon MSH2-MSH6 binding, specifically addressing the open or stacked nature of the junction upon complexation with MSH2-MSH6.To characterize the MSH4-MSH5 binding interactions with Holliday Junctions, our efforts are currently focused on cloning S. cerevisiae msh4 and msh5 genes for over-expression in E. coli. We have successfully cloned the msh4 gene and msh5 genes from the wild type yeast genome and inserted these two genes separately into the two multiple cloning sites on the pCDFDuet-1 vector. Optimal conditions for expressing MSH4-MSH5 heterodimers are being explored and will be presented.