Inhibition of the Escherichia coli replication cycle by a mutant RecA protein
Abstract: A conserved [K/R]x[K/R] motif is found at the subunit-subunit interface in bacterial RecA protein filaments. In the Escherichia coli RecA protein, this motif is made up of residues K248 and K250. Both of these residues have a role in ATP hydrolysis (in trans across the interface) and in the transmission of conformational information across the interface. A K250R mutation creates a RecA protein that promotes both ATP hydrolysis and DNA-strand exchange at a six-fold lower rate than the wild type protein. E. coli strains overexpressing this mutant RecA protein also grow much slower than wild type strains. Suppressor mutations appear quickly, and most suppressor mutations inactivate RecA (leading to high levels of UV sensitivity). One suppressor mutation, A11V, did not inactivate the RecA protein, but instead produced a RecA variant that allowed normal rates of growth under normal growth conditions, while partially restoring UV resistance. We found that this suppressor mutation has no effect on the rate of ATP hydrolysis of the mutant RecA K250R protein, but has slightly better strand exchange activity than RecA K250R. We also found that RecA K250R/A11V has a higher rate of disassembly than RecA K250R, showing that this suppression mutation allowed the double mutant RecA nucleoprotein filament to be more dynamic. Our hypothesis is that the [KR]x[KR] motif, especially RecA residue K250 is directly involved in RecA functions. Through mutational studies, we can get a better understanding of the role of this residue in RecA-catalyzed reactions.