Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η*
Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η*
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保守酪氨酸 52 处的氨基酸取代改变人类 DNA 聚合酶 η* 的保真度和旁路效率
DOI：
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发表时间：
2003
影响因子：
4.8
通讯作者：
L. Loeb
中科院分区：
文献类型：
--
作者：
E. Glick;Janice S. Chau;K. Vigna;S. McCulloch;E. Adman;T. Kunkel;L. Loeb
DNA聚合酶η(PolDNA聚合酶)是一类能够复制含有受损核苷酸的DNA聚合酶的新成员。这些聚合酶在复制未改变的DNA模板时非常容易出错。我们通过在人DNA聚合酶η(HPOLη)指状域中引入高度保守的氨基酸Tyr-52的替换来分析旁路效率与DNA合成保真度之间的关系。大多数Tyr-52的替代导致了紫外线相关损伤旁路的减少，这是通过在高紫外线剂量下拯救紫外线敏感的酵母细胞的能力来衡量的。对于大多数突变体来说，旁路能力的降低与聚合活性的降低是平行的。有趣的是，Hpol1ηY52E突变体表现出比聚合活性更大的旁路效率下降。旁路效率降低的同时，互补核苷酸的掺入比非互补核苷酸增加了11倍。DNA合成的保真度，通过在体外复制有间隙的M13 DNA模板来衡量，也提高了15倍；这种增强是由于相对频繁的转变的减少，以及颠换的大幅减少。我们的研究表明，活性部位内的氨基酸取代提高了HPOLDNA聚合酶合成的保真度，HPOLDNA聚合酶是最不准确的η聚合酶之一，支持这样的假设，即即使是容易出错的DNA聚合酶也在碱基选择中发挥作用。
DNA polymerase η (Polη) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase η (hPolη) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPolη Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to non-complementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPolη, one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.
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影响因子：
16
作者：
Trincao, J;Johnson, RE;Aggarwal, AK
通讯作者：
Aggarwal, AK
DOI：
10.1016/0027-5107(91)90157-j
发表时间：
1991-09-01
期刊：
MUTATION RESEARCH
影响因子：
--
作者：
AMES, BN;GOLD, LS
通讯作者：
GOLD, LS
影响因子：
11.2
作者：
L. Loeb
通讯作者：
L. Loeb
影响因子：
56.9
作者：
Johnson, RE;Kondratick, CM;Prakash, L
通讯作者：
Prakash, L
酿酒酵母 DNA 聚合酶 eta 绕过胸腺嘧啶-胸腺嘧啶二聚体的准确性。
DOI：
10.1073/pnas.97.7.3094
发表时间：
2000
影响因子：
11.1
作者：
Washington,MT;Johnson,RE;Prakash,S;Prakash,L
通讯作者：
Prakash,L