| Abstrakt: |
The ability of a DNA polymerase to replicate DNA beyond a mismatch containing a DNA lesion during postlesion DNA synthesis (PLS) can be a contributing factor to mutagenesis. In this study, we investigate the ability of Dpo4, a Y-family DNA polymerase from Sulfolobus solfataricus, to perform PLS beyond the pro-mutagenic DNA adducts O6-benzylguanine (O6-BnG) and O6-methylguanine (O6-MeG). Here, O6-BnG and O6-MeG were paired opposite artificial nucleosides that were structurally altered to systematically test the influence of hydrogen bonding and base pair size and shape on O6-alkylguanine PLS. Dpo4-mediated PLS was more efficient past pairs containing Benzi than pairs containing the other artificial nucleoside probes. Based on steady-state kinetic analysis, frequencies of mismatch extension were 7.4 × 10–3and 1.5 × 10–3for Benzi:O6-MeG and Benzi:O6-BnG pairs, respectively. Correct extension was observed when O6-BnG and O6-MeG were paired opposite the smaller nucleoside probes Benzi and BIM; conversely, Dpo4 did not extend past the larger nucleoside probes, Peri and Per, placed opposite O6-BnG and O6-MeG. Interestingly, Benzi was extended with high fidelity by Dpo4 when it was paired opposite O6-BnG and O6-MeG but not opposite G. These results indicate that hydrogen bonding is an important noncovalent interaction that influences the fidelity and efficiency of Dpo4 to perform high-fidelity O6-alkylguanine PLS. |
