HMSH4-hMSH5 Adenosine Nucleotide Processing and Interactions with Homologous Recombination Machinery

Christoph Schmutte,Richard Fishel,Kang-Sup Shim,Samir Acharya,Timothy Snowden

doi:10.1074/jbc.m704060200

Abstract

We have previously demonstrated that the human heterodimeric meiosis-specific MutS homologs, hMSH4-hMSH5, bind uniquely to a Holliday Junction and its developmental progenitor (Snowden, T., Acharya, S., Butz, C., Berardini, M., and Fishel, R. (2004) Mol. Cell 15, 437-451). ATP binding by hMSH4-hMSH5 resulted in the formation of a sliding clamp that dissociated from the Holliday Junction crossover region embracing two duplex DNA arms. The loading of multiple hMSH4-hMSH5 sliding clamps was anticipated to stabilize the interaction between parental chromosomes during meiosis double-stranded break repair. Here we have identified the interaction region between the individual subunits of hMSH4-hMSH5 that are likely involved in clamp formation and show that each subunit of the heterodimer binds ATP. We have determined that ADP-->ATP exchange is uniquely provoked by Holliday Junction recognition. Moreover, the hydrolysis of ATP by hMSH4-hMSH5 appears to occur after the complex transits the open ends of model Holliday Junction oligonucleotides. Finally, we have identified several components of the double-stranded break repair machinery that strongly interact with hMSH4-hMSH5. These results further underline the function(s) and interactors of hMSH4-hMSH5 that ensure accurate chromosomal repair and segregation during meiosis.

Highlights

MutS homologs (MSH)2 have been identified in most organisms examined
Quantitation was displayed relative to the known interactions between fulllength hMSH4 and hMSH5 (Intrel)
We demonstrate that full-length hMSH4 interacts strongly with hMSH5 either as an in vitro transcription-translation (IVTT)-hMSH4 with GST-hMSH5 (Fig. 1A, lane 1) or a GST-hMSH4 with IVTT-hMSH5 (Fig. 1B, lane 1)

Summary

Introduction

MutS homologs (MSH) have been identified in most organisms examined. Bacteria may contain one or two MSH proteins, whereas the yeast Saccharomyces cerevisiae has been found to contain six MSH proteins (MSH1–MSH6), five of which are conserved in human (hMSH2– hMSH6) (for review see Refs. 1 and 2). The Escherichia coli MutS functions as an asymmetric homodimer This fundamental asymmetric nature appears conserved in the five human MSH proteins that form three heterodimers, hMSH2-hMSH3, hMSH2-hMSH6, and MSH4MSH5 [3,4,5]. Genetic studies in S. cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, and Mus musculus suggest that MSH4 and MSH5 are not involved in MMR but are required for normal chromosomal segregation and the production of viable gametes during meiosis [17,18,19,20,21,22,23,24,25]. Such dHoJo’s have been observed in vivo during meiosis in yeast [31]

Methods

Results

Conclusion