Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis.
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 λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β 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 λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ 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 λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.
Brown
Jessica A
JA
Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Pack
Lindsey R
LR
Sanman
Laura E
LE
Suo
Zucai
Z
D020816:Amino Acid Motifs / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D003854:Deoxyribonucleotides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D013379:Substrate Specificity
D003854:Deoxyribonucleotides; D004247:DNA; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta