Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance.
Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance.
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DOI：
10.1074/jbc.m115.662841
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发表时间：
2015-09-04
期刊：
影响因子：
--
通讯作者：
Gardner AF
中科院分区：
文献类型：
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作者：
Schermerhorn KM;Gardner AF
背景：D家族DNA聚合酶（polD）在除泉古菌外的大多数古菌中是重要的复制酶。结果：我们报告了polD核苷酸掺入、错配识别和3′-5′核酸外切酶水解的详细动力学特征。结论：尽管进化上存在分歧，但polD动力学途径与其他DNA聚合酶家族有相似之处。意义：这项工作有助于统一我们对DNA聚合酶功能的理解。除了泉古菌分支外，D家族DNA聚合酶（polD）是古菌中主要的复制聚合酶，并且与其他DNA聚合酶家族几乎没有序列同源性。在这里，我们报告了一个详细的动力学分析的核苷酸掺入和核酸外切酶活性的D家族DNA聚合酶从Thermococcus sp.9 °N。进行预稳态单周转核苷酸掺入试验，以获得正确核苷酸掺入、不正确核苷酸掺入和核糖核苷酸掺入的动力学参数kpol和Kd。正确的核苷酸掺入动力学显示，与A、B、C、X和Y家族的DNA聚合酶相比，其最大聚合速率相对较慢（kpol <2.5 s-1），尤其是核苷酸结合紧密（Kd（dNTP）<1.7 μm）。此外，预稳态核苷酸掺入测定揭示，polD主要通过降低核苷酸结合亲和力来防止不正确的核苷酸和核糖核苷酸的掺入。利用9°N polD野生型的预稳态单转换试验检测了Mg 2+和Mn 2+存在下3′-5′核酸外切酶的水解活性。有趣的是，用Mn 2+取代Mg 2+，水解速率加快了40倍以上（kexo ≥110 s−1 vs ≥2.5 s−1）。在核酸外切酶水解活性中Mn 2+优于Mg 2+是polD家族特有的性质。在这项工作中进行的动力学分析提供了关键的洞察polD采用的机制，准确和有效地复制古细菌基因组。此外，尽管polD的独特性质，这项工作表明，保守的聚合酶动力学途径存在于所有已知的DNA聚合酶家族。
Background: Family D DNA polymerase (polD) is important for replication in most archaea, excluding Crenarchaeota. Results: We report a detailed kinetic characterization of polD nucleotide incorporation, mismatch discrimination, and 3′-5′ exonuclease hydrolysis. Conclusion: Despite evolutionary divergence, polD kinetic pathways share similarities to other DNA polymerase families. Significance: This work contributes to unifying our understanding of DNA polymerase function. Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, kpol and Kd, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (kpol ∼2.5 s−1) and especially tight nucleotide binding (Kd(dNTP) ∼1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg2+ and Mn2+. Interestingly, substituting Mn2+ for Mg2+ accelerated hydrolysis rates >40-fold (kexo ≥110 s−1 versus ≥2.5 s−1). Preference for Mn2+ over Mg2+ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families.