Escherichia coli DNA polymerase II can efficiently bypass 3,N4-ethenocytosine lesions in vitro and in vivo

Escherichia coli DNA polymerase II (pol-II) is a highly conserved protein that appears to have a role in replication restart, as well as in translesion synthesis across specific DNA adducts under some conditions. Here, we have investigated the effects of elevated expression of pol-II (without concomitant SOS induction) on translesion DNA synthesis and mutagenesis at 3,N4-ethenocytosine (ɛC), a highly mutagenic DNA lesion induced by oxidative stress as well as by exposure to industrial chemicals such as vinyl chloride. In normal cells, survival of transfected M13 single-stranded DNA bearing a single ɛC residue (ɛC-ssDNA) is about 20% of that of control DNA, with about 5% of the progeny phage bearing a mutation at the lesion site. Most mutations are C → A and C → T, with a slight predominance of transversions over transitions. In contrast, in cells expressing elevated levels of pol-II, survival of ɛC-ssDNA is close to 100%, with a concomitant mutation frequency of almost 99% suggesting highly efficient translesion DNA synthesis. Furthermore, an overwhelming majority of mutations at ɛC are C → T transitions. Purified pol-II efficiently catalyzes translesion synthesis at ɛC in vitro, accompanied by high levels of mutagenesis with the same specificity. These results suggest that the observed in vivo effects in pol-II over-expressing cells are due to pol-II-mediated DNA synthesis. Introduction of mutations in the carboxy terminus region (β interaction domain) of polB eliminates in vivo translesion synthesis at ɛC, suggesting that the ability of pol-II to compete with pol-III requires interaction with the β processivity subunit of pol-III. Thus, pol-II can compete with pol-III for translesion synthesis.