Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

被引:38
|
作者
Brown, Jessica A. [1 ,2 ]
Pack, Lindsey R. [1 ]
Sanman, Laura E. [1 ]
Suo, Zucai [1 ,2 ,3 ,4 ,5 ]
机构
[1] Ohio State Univ, Dept Biochem, Columbus, OH 43210 USA
[2] Ohio State Univ, Ohio State Biochem Program, Columbus, OH 43210 USA
[3] Ohio State Univ, Ohio State Biophys Program, Columbus, OH 43210 USA
[4] Ohio State Univ, Mol Cellular & Dev Biol Program, Columbus, OH 43210 USA
[5] Ohio State Univ, Ctr Comprehens Canc, Columbus, OH 43210 USA
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
Base excision repair; X-family DNA polymerase; DNA polymerase lambda; DNA polymerase beta; Pre-steady state kinetics; BASE-EXCISION-REPAIR; PURIFIED HUMAN PROTEINS; LYASE ACTIVITY; STRAND MISALIGNMENT; DEPENDENT REPAIR; MAMMALIAN-CELLS; IN-VITRO; DAMAGE; REQUIREMENT; PROTECTS;
D O I
10.1016/j.dnarep.2010.09.005
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
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 lambda (Pol lambda), an enzyme proposed to support the known BER DNA polymerase beta (Pot beta), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol lambda incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol lambda, Pol beta 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 lambda, and this downregulation of fidelity was controlled by its nonenzymatic N-terminal domains. Overall, Pol lambda was up to 160-fold more error-prone than Pol beta, thereby suggesting Pol lambda would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol lambda 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 lambda and Pol beta would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol lambda, like Pot beta, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol lambda may play a role in long-patch BER. (C) 2010 Elsevier B.V. All rights reserved.
引用
收藏
页码:24 / 33
页数:10
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