Mechanism of mycobacterial recombination mediator proteins function in homologous recombination and strand annealing (735.12)

Abstract

Recombination mediator proteins (RMPs) are important for genome stability in all organisms and support two reactions: the recombinase binding to single‐stranded (ss)DNA to initiate homologous recombination (HR) and strand annealing. Both reactions are inhibited by ssDNA binding proteins (e.g. gp32, SSB, RPA). RMPs promote DNA transfer from SSB to recombinase or to anneal DNA. In spite of two ubiquitous functions, RMPs lack sequence and structural homology. Previously, we demonstrated that E. coli RMPs, RecOR, do not dismiss SSB from ssDNA, but form the complex with SSB altering the conformation of bound ssDNA. RecO binds SSB and the complex promotes DNA annealing or RecA loading in the absence or presence of RecR, respectively. Surprisingly, mycobacterial RecO does not interact with SSB. Yet, both proteins form complex on the shared ssDNA substrate in both pathways.In mycobacteria, RecFOR deletion reduces clastogens resistance to the same degree as a deletion of the major homologous recombinase RecA. Chromosomal DNA break repair assay surprisingly revealed that all three proteins are involved in both pathways: HR and annealing. Here we will describe biochemical studies of DNA annealing and strand exchange reactions supported by mycobacterial RecFOR. The selection of one or the other pathway is dictated by the availability of recombinase or complimentary strand and the stoichiometry of the DNA‐protein complexes. We believe that the intermediate complex of RMP with SSB and ssDNA represents a common critical step in reactions supported by all RMPs during HR and strand annealing DNA repair pathways in all organisms.