DNA binding site II is required for RAD51 recombinogenic activity in Arabidopsis thaliana

Abstract

Homologous recombination is a major pathway for the repair of DNA double strand breaks, essential both to maintain genomic integrity and to generate genetic diversity. Mechanistically, homologous recombination involves the use of a homologous DNA molecule as a template to repair the break. In eukaryotes, the search for and invasion of the homologous DNA molecule is carried out by two recombinases, RAD51 in somatic cells and RAD51 and DMC1 in meiotic cells. During recombination, the recombinases bind overhanging single-stranded DNA ends to form a nucleoprotein filament, which is the active species in promoting DNA invasion and strand exchange. RAD51 and DMC1 carry two major DNA binding sites - essential for nucleofilament formation and DNA strand exchange, respectively. Here, we show that the function of RAD51 DNA binding Site II is conserved in the plant, Arabidopsis. Mutation of three key amino acids in Site II does not affect RAD51 nucleofilament formation but inhibits its recombinogenic activity, analogous to results from studies of the yeast and human proteins. We further confirm that recombinogenic function of RAD51 DNA binding site II is not required for meiotic DSB repair when DMC1 is present. The Arabidopsis RAD51-II3A separation of function mutant shows a dominant negative phenotype, pointing to distinct biochemical properties of eukaryotic RAD51 proteins. Summary blurbThe activity promoted by RAD51 DNA binding site II is not required for nucleofilament formation, but is essential for subsequent invasion and strand exchange activity of RAD51 in Arabidopsis thaliana.