Efficiency and fidelity of human DNA polymerases ? and ? during gap-filling DNA synthesis.

ABSTRACT: The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase ? (Pol ?), an enzyme proposed to support the known BER DNA polymerase ? (Pol ?), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol ? incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol ?, Pol ? did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol ?, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol ? was up to 160-fold more error-prone than Pol ?, thereby suggesting Pol ? would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol ? and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol ? and Pol ? would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol ?, like Pol ?, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol ? may play a role in long-patch BER.

SUBMITTER: Brown JA 

PROVIDER: S-EPMC3065367 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature