Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2147730 | Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis | 2006 | 13 Pages |
Abstract
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 (ÉC), 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 ÉC residue (ÉC-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 â A and C â T, with a slight predominance of transversions over transitions. In contrast, in cells expressing elevated levels of pol-II, survival of ÉC-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 ÉC are C â T transitions. Purified pol-II efficiently catalyzes translesion synthesis at ÉC 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 ÉC, 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.
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Authors
Abu Amar M. Al Mamun, M. Zafri Humayun,