Person:

Kath, James Evon

Translesion synthesis (TLS) alleviates replication stalling at DNA lesions. Bypass of lesions by specialized translesion DNA polymerases involves exchange with high-fidelity replicative polymerases. As a consequence of their lesion bypass activity, TLS polymerases are mutagenic, requiring careful regulation of polymerase selection. In this dissertation, I describe a single-molecule reconstitution of polymerase exchange and lesion bypass. Using Escherichia coli polymerases as a model system, I have determined that the dimeric processivity clamp can simultaneously bind a replicative polymerase and a translesion polymerase, facilitating rapid exchange during synthesis and lesion bypass. Overlapping sets of polymerase-clamp interactions additionally allow the TLS polymerase Polymerase IV to displace the replicative polymerase Polymerase III. I finally describe the observation of single Polymerase IV molecules in living cells and initial efforts to determine their localization and dynamics during TLS.

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the β sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the β clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.