An antimorphic mutant of Brh2 resulting from a conservative amino acid change within the BRC element

Brh2, the streamlined BRCA2 family protein from Ustilago maydis, directs homologous recombination by modulating Rad51 filament dynamics through a single BRC element. Here, we describe an unusual antimorphic mutation within the BRC β-hairpin of Brh2, in which the pivotal phenylalanine residue in the sequence mimicking the Rad51 polymerization motif is replaced by methionine. This methionine-for-phenylalanine substitution in the BRC of Brh2 results in a DNA repair defect more severe than the null allele, while the cognate methionine-for-phenylalanine substitution in Rad51's polymerization motif supports normal function. The dichotomy suggests that the switch to methionine from phenylalanine exerts distinct structural effects depending on molecular context. Suppression of the antimorphic phenotype by a secondary mutation (T296A) destabilizing the β-hairpin implies that the deleterious activity of the mutant variant requires an intact BRC fold. Relocation of the mutant BRC to an ectopic site within Brh2 mitigates the defect. This indicates there is positional dependence and hierarchical use of BRC modules within the protein. Biochemical assays with BRC polypeptides demonstrate that the BRC^F294M variant is reduced in ability to form stable complexes with free Rad51. However, analysis shows BRC^F294M engages Rad51-ssDNA filaments aberrantly promoting atypical aggregation rather than normal filament dynamics. These findings suggest a mechanistic basis for this antimorphic BRC mutation and highlight the critical influence of BRC structural context in governing Rad51 filament dynamics and DNA repair proficiency.