Direct Visualization of Translesion DNA Synthesis Polymerase IV at the Replisome

Abstract

In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these TLS DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the b sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the b clamp but not its catalytic activity, and is reinforced by the presence of the g subunit of the b clamp-loading DnaX complex in the DNA polymerase III holoenzyme.