Analysis of the stimulation of DNA polymerase V of Escherichia coli by processivity proteins.

Abstract

Bypass of replication-blocking lesions in Escherichia coli is carried out by DNA polymerase V (UmuC) in a reaction that requires UmuD', RecA, and single-strand DNA-binding protein (SSB). The activity of this four-component basic bypass system is a low-fidelity and low-processivity activity. Addition of the processivity subunits of pol III, the beta subunit sliding DNA clamp, and the five-subunit gamma complex clamp loader increased the rate of translesion replication approximately 3-fold. This stimulation was specific to the lesion bypass step, with no effect on the initiation of synthesis by pol V. The beta subunit and gamma complex increased the processivity of pol V from 3 to approximately 14-18 nucleotides, providing a mechanistic basis for their stimulatory effect. Stimulation of bypass was observed over a range of RecA and SSB concentrations. ATPgammaS, which strongly inhibits translesion replication by pol V, primarily via inhibition of the initiation stage, caused the same inhibition also in the presence of the processivity proteins. The in vivo role of the processivity proteins in translesion replication was examined by assaying UV mutagenesis. This was done in a strain carrying the dnaN59 allele, encoding a temperature-sensitive beta subunit. When assayed in an excision repair-defective background, the dnaN59 mutant exhibited a level of UV mutagenesis reduced up to 3-fold compared to that of the isogenic dnaN(+) strain. This suggests that like in the in vitro system, the beta subunit stimulates lesion bypass in vivo.