Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.
Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.
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DOI：
10.1021/tx5002755
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发表时间：
2014-10-20
影响因子：
4.1
通讯作者：
Choi JY
中科院分区：
文献类型：
--
作者：
Kim J;Song I;Jo A;Shin JH;Cho H;Eoff RL;Guengerich FP;Choi JY
DNA聚合酶（pol）1是参与跨损伤合成（TLS）的Y家族聚合酶中最容易出错的。Pol 1可以绕过各种DNA损伤，例如，N2-乙基（Et）G、O 6-甲基（Me）G、8-氧代-7，8-二氢鸟嘌呤（8-oxoG）和脱碱基位点，但通常保真度较低。我们使用重组pol 1（残基1-445）蛋白和含有G、N2-EtG、O 6-MeG、8-oxoG或脱碱基位点的DNA模板，评估了六种已报道的人类pol 1遗传变异对其TLS性质的生物化学效应。Δ1-25变体，其是由起始密码子变体产生的25个残基的N-末端截短（c.3G > A），也是以前错误分配的野生型，在Mg 2+存在下表现出比野生型高得多的聚合酶活性（但不含Mn 2+），这与其稳态动力学数据相一致，显示核苷酸掺入相对模板（仅用Mg 2+）的kcat/Km增加了10倍。缺少已知与进入的核苷酸相互作用的R96残基的R96 G变体丧失了其大部分聚合酶活性，这与动力学数据一致，所述动力学数据显示对于具有Mg 2+或Mn 2+的相对模板，核苷酸掺入的kcat/Km降低5至72倍，除了具有Mn 2+的相对N2-EtG（显示dCTP掺入增加9倍）。Δ1-25变体与DNA的结合比野生型紧密20至29倍（与Mg 2+），但R96 G变体与DNA的结合比野生型紧密2倍。与0.15 mM Mn 2+相比，野生型的DNA结合亲和力（而非Δ1-25变体的DNA结合亲和力）是Mg 2+的2.7倍。结果表明，R96 G变异严重削弱了poll的大部分Mg 2+和Mn 2+依赖性TLS能力，而Δ1-25变异选择性地且显著地增强了poll的Mg 2+依赖性TLS能力，强调了这些poll遗传变异的潜在翻译重要性，例如，TLS、突变和癌症对遗传毒性致癌物易感性的个体差异。
DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N2-ethyl(Et)G, O6-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1–445) proteins and DNA templates containing a G, N2-EtG, O6-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg2+ (but not with Mn2+), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg2+). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg2+ or Mn2+, except for that opposite N2-EtG with Mn2+ (showing a 9-fold increase for dCTP incorporation). The Δ1–25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg2+), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1–25 variant, was ∼7-fold stronger with 0.15 mM Mn2+ than with Mg2+. The results indicate that the R96G variation severely impairs most of the Mg2+- and Mn2+-dependent TLS abilities of pol ι, whereas the Δ1–25 variation selectively and substantially enhances the Mg2+-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens.
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影响因子：
4.8
作者：
Choi, JY;Guengerich, FP
通讯作者：
Guengerich, FP
影响因子：
4.8
作者：
Choi, JY;Guengerich, FP
通讯作者：
Guengerich, FP
影响因子：
4.8
作者：
Choi, Jeong-Yun;Chowdhury, Goutam;Guengerich, F. Peter
通讯作者：
Guengerich, F. Peter
影响因子：
4.1
作者：
Ketkar A;Zafar MK;Maddukuri L;Yamanaka K;Banerjee S;Egli M;Choi JY;Lloyd RS;Eoff RL
通讯作者：
Eoff RL
影响因子：
10.3
作者：
Kennedy, DO;Agrawal, M;Santella, RM
通讯作者：
Santella, RM