Pre-steady-state kinetic studies of the fidelity of Sulfolobus solfataricus P2 DNA polymerase IV
Pre-steady-state kinetic studies of the fidelity of Sulfolobus solfataricus P2 DNA polymerase IV
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DOI：
10.1021/bi0357457
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发表时间：
2004-02-24
期刊：
影响因子：
2.9
通讯作者：
Suo, Z
中科院分区：
文献类型：
--
作者：
Fiala, KA;Suo, Z
solfataricus P2 DNA聚合酶IV （Dpo4）是一种耐热古细菌酶，是易出错和绕过病变的y家族成员。在本文中，y家族聚合酶Dpo4的保真度首次通过在37度下将单个核苷酸掺入未损坏的DNA底物21/41-mer的预稳态动力学分析来确定。我们评估了所有16种可能的核苷酸结合的单次转换（Dpo4在DNA上的摩尔过量）饱和动力学。Dpo4的保真度估计在10(-3)-10（-4）之间。有趣的是，正确核苷酸（70-230 muM）的基态结合亲和力比复制性DNA聚合酶弱10-50倍。这种低亲和力与Dpo4与结合核苷酸之间缺乏相互作用是一致的，这可以从Dpo4、DNA和匹配核苷酸的晶体结构中看出。错误的核苷酸对Dpo4的亲和力比正确的核苷酸弱2-10倍。有趣的是，当不匹配的dCTP与嘧啶模板碱基结合时，它具有与匹配的核苷酸相似的亲和力，该碱基的两侧是一个5'模板鸟嘌呤。进入的dCTP可能会跳过第一个可用的模板碱基和5'-模板鸟嘌呤碱基对，正如在Dpo4， DNA和不匹配的核苷酸的晶体结构中观察到的那样。不考虑5'-模板碱基，错配结合率比匹配核苷酸的结合率低2-3个数量级，这是对Dpo4保真度的主要贡献。
Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a thermostable archaeal enzyme and a member of the error-prone and lesion-bypass Y-family. In this paper, for the first time, the fidelity of a Y-family polymerase, Dpo4, was determined using pre-steady-state kinetic analysis of the incorporation of a single nucleotide into an undamaged DNA substrate 21/41-mer at 37 degreesC. We assessed single-turnover (with Dpo4 in molar excess over DNA) saturation kinetics for all 16 possible nucleotide incorporations. The fidelity of Dpo4 was estimated to be in the range of 10(-3)-10(-4). Interestingly, the ground-state binding affinity of correct nucleotides (70-230 muM) is 10-50-fold weaker than those of replicative DNA polymerases. Such a low affinity is consistent with the lack of interactions between Dpo4 and the bound nucleotides as revealed in the crystal structure of Dpo4, DNA, and a matched nucleotide. The affinity of incorrect nucleotides for Dpo4 is similar to2-10-fold weaker than that of correct nucleotides. Intriguingly, the mismatched dCTP has an affinity similar to that of the matched nucleotides when it is incorporated against a pyrimidine template base flanked by a 5'-template guanine. The incoming dCTP likely skips the first available template base and base pairs with the 5'-template guanine, as observed in the crystal structure of Dpo4, DNA, and a mismatched nucleotide. The mismatch incorporation rates, regardless of the 5'-template base, were similar to2-3 orders of magnitude slower than the incorporation rates for matched nucleotides, which is the predominant contribution to the fidelity of Dpo4.