Dissection of biophysical properties and biological activities of Bet/RecT family DNA recombination proteins

Abstract

Single strand DNA annealing proteins (SSAPs) of viral/phage origin play a variety of roles in homologous recombination-based DNA repair and genetic exchange processes in prokaryotes. Bet from bacteriophage lambda and RecT from Escherichia coli are the prototypical SSAPs. Bet/RecT family SSAP proteins putatively share a conserved multimeric organization and arrangement of secondary structural units. Here, we use a combination of biophysical and biological approaches to dissect structure-activity relationships within a diverse selection of SSAP proteins, including: E. coli RecT; Lambda-Bet; s065 from the SXT genetic element of Vibrio cholerae (SXT-Bet) and Bet from Laribacter hongkongensis (LHK-Bet). Single nucleotide alteration and double strand DNA deletion activities were investigated using E. coli reporter systems. We show that Bet/RecT SSAP proteins that have been rationally-truncated at the N-terminus, but not at the C-terminus, exhibit DNA recombination activities. The removal of specific secondary structural units from Bet/RecT SSAPs profoundly affects protein multimer formation in a complex manner. Binding assays indicate that DNA recombination activities are not directly related to single strand DNA binding abilities. Our results suggest that a complex interplay of protein-protein and protein-nucleotide interactions underlie DNA recombination activities in Bet/RecT family SSAP proteins.