Characterization of a potent dominant negative mutant variant of Rad51 in Ustilago maydis

• Published in: DNA repair Vol. 78; pp. 91 - 101
• Main Authors: Sutherland, Jeanette H., Holloman, William K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2019
• Subjects: Alleles; Amino Acid Sequence; BRCA2; DNA Damage; DNA repair; Dominant negative; Homologous recombination; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation; Phenotype; Rad51; Rad51 Recombinase - chemistry; Rad51 Recombinase - genetics; Rad51 Recombinase - metabolism; Ustilago - enzymology; Rad51; DEB; ss; UV; MMS; Homologous recombination; Dominant negative; HR; BRCA2; DNA repair
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2019.04.003

•Rad51 variant D161Y confers a strong dominant negative effect on DNA repair.•Modeling suggests protomer-protomer interference as the basis of the phenotyp.•Additional mutational changes in Rad51 abrogate the dominant negative effect.•D161Y variant interferes with Rad51 nucleoprotein filament formation and stability. Rad51 serves to maintain and protect integrity of the genome through its actions in DNA repair and replication fork protection. The active form of Rad51 is a nucleoprotein filament consisting of chains of protomer units arranged linearly along single-stranded DNA. In a mutant screen using Ustilago maydis as an experimental system we identified a novel variant of Rad51, in which an amino acid change near the protomer–protomer interaction interface confers a strong trans dominant inhibitory effect on resistance to DNA damaging agents and proficiency in homologous recombination. Modeling studies of the mutated residue D161Y suggested that steric interference with surrounding residues was the likely cause of the inhibitory effect. Changes of two nearby residues, predicted from the modeling to minimize steric clashes, mitigated the inhibition of DNA repair. Direct testing of purified Rad51D161Y protein in defined biochemical reactions revealed it to be devoid of DNA-binding activity itself, but capable of interfering with Rad51WT in formation and maintenance of nucleoprotein filaments on single-stranded DNA and in DNA strand exchange. Rad51D161Y protein appears to be unable to self-associate in solution and defective in forming complexes with the U. maydis BRCA2 ortholog.