Bridging the gap: a family of novel DNA polymerases that replicate faulty DNA.
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ABSTRACT:
SUBMITTER: Johnson RE
PROVIDER: S-EPMC34254 | biostudies-literature | 1999 Oct
REPOSITORIES: biostudies-literature
Publications
Bridging the gap: a family of novel DNA polymerases that replicate faulty DNA.
Johnson R E RE Washington M T MT Prakash S S Prakash L L
Proceedings of the National Academy of Sciences of the United States of America 19991001 22
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Project description: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.
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