Tolerance of Base Pair Size and Shape in Postlesion DNA Synthesis

Abstract

The influence of base pair size and shape on the fidelity of DNA polymerase-mediated extension past lesion-containing mispairs was examined. Primer extension analysis was performed with synthetic nucleosides paired opposite the pro-mutagenic DNA lesion <i>O</i>⁶-benzylguanine (<i>O</i>⁶-BnG). These data indicate that the error-prone DNA polymerase IV (Dpo4) inefficiently extended past the larger Peri:<i>O</i>⁶-BnG base pair, and in contrast, error-free extension was observed for the smaller BIM:<i>O</i>⁶<i>-</i>BnG base pair. Steady-state kinetic analysis revealed that Dpo4 catalytic efficiency was strongly influenced by the primer:template base pair. Compared to the C:G pair, a 1.9- and 79 000-fold reduction in Dpo4 efficiency was observed for terminal C:<i>O</i>⁶-BnG and BIM:G base pairs respectively. These results demonstrate the impact of geometrical size and shape on polymerase-mediated mispair extension