Determinants governing BRC function evaluated by mutational analysis of Brh2 in Ustilago maydis

Abstract

BRC is a short evolutionarily conserved sequence motif generally arranged in multiple tandem repeats that is present as a defining feature in members of the BRCA2 tumor suppressor protein family. From crystallographic studies of a co-complex, the human BRC4 was found to form a structural element that interacts with RAD51, a key component in the DNA repair machinery directed by homologous recombination. The BRC is distinguished by two tetrameric sequence modules with characteristic hydrophobic residues separated by an intervening spacer region marked by certain highly conserved residues forming a hydrophobic surface for interaction with RAD51. It is present as a single copy in Brh2 of Ustilago maydis, the only reported example of a fungal BRCA2 ortholog. By comparative sequence analysis, examples of BRCA2 orthologs were identified in other fungal phyla, some of which featured multiple tandem repeats like those found in mammals. An expeditious biological assay system was developed for evaluating the two-tetramer module model and assessing the importance of particular conserved amino acid residues of BRC contributing to Brh2 functionality in DNA repair. This work was aided by the finding that the human BRC4 repeat could substitute completely for the endogenous BRC element in Brh2, while the human BRC5 repeat could not. In a survey of point mutations of certain residues, certain BRC mutant variants termed antimorphs were identified that caused a DNA repair phenotype more severe than the null.