Approaches to Understanding the Mediator Function of Brh2 in Ustilago maydis

Abstract

Primary components of the homologous recombination pathway in eukaryotes include Rad51 whose function is to search for DNA sequence homology and promote strand exchange, its mediator BRCA2, and Dss1, a key regulator of BRCA2. We seek to understand the role of BRCA2 in governing the activity of Rad51 and to learn how BRCA2 function is regulated by Dss1. We use the microbe Ustilago maydis as a model system for experimentation because it has a well-conserved BRCA2-homolog, Brh2, and is amenable to biochemical and molecular genetic manipulations and analysis. The powerful attributes of this system open the way for gaining insight into BRCA2's molecular mechanism through avenues not immediately approachable in the vertebrate systems. Here we provide protocols for preparing Brh2, Dss1, and Rad51 as reagents for use in biochemical assays to monitor function and present methods for transposon-based mutational analysis of Brh2 for use in genetic dissection of function.