DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli

Abstract

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.