Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.
Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.
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DOI：
10.1016/j.jmb.2016.06.008
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发表时间：
2016-07-17
影响因子：
5.6
通讯作者：
Connolly BA
中科院分区：
文献类型：
--
作者：
Abellón-Ruiz J;Waldron KJ;Connolly BA
古细菌家族d DNA聚合酶（Pol-D）包括一个小的（DP1）校对亚基和一个大的（DP2）聚合酶亚基。Pol-D是研究最少的聚合酶家族之一，本出版物研究了来自fulgidus （Afu Pol-D）的酶。DP2的c端区域包含两个保守的半胱氨酸簇，它们的作用是通过定点突变来研究的。离C端最近的簇对于聚合酶的活性是必不可少的，并且半胱氨酸被证明可以作为单一的、临界的Zn2 +离子的配体。离C端最远的半胱氨酸不需要活性，这些氨基酸的作用尚未确定。此外，研究表明，Afu Pol-D活性被模板链次黄嘌呤减慢，扩展了先前证明尿嘧啶抑制的结果。次黄嘌呤的抑制作用弱于尿嘧啶。分离的DP2具有可测量的聚合酶活性，研究将脱胺碱基结合位点定位在该亚基上。尿嘧啶和次黄嘌呤“in trans”减缓Afu Pol-D，也就是说，复制的DNA链可以被复制叉的另一条链上的去胺化碱基抑制。体外测定的Afu Pol-D的错误率为0.24 × 10−5，这是一种被认为在古细菌中进行基因组复制的聚合酶的典型错误率。删除3 ‘ -5 ’校对外切酶活性使保真度降低了两倍。本出版物中提出的结果大大增加了我们对Pol-D的了解。古细菌DNA聚合酶d结合锌，这是活性所必需的。古生菌DNA聚合酶d被尿嘧啶和次黄嘌呤抑制。脱氨基碱基的抑制作用在顺式和反式中都存在。脱胺碱基由大（聚合酶）亚基感应。聚合酶d具有高保真度，与基因组复制兼容。
Archaeal family-D DNA polymerases (Pol-D) comprise a small (DP1) proofreading subunit and a large (DP2) polymerase subunit. Pol-D is one of the least studied polymerase families, and this publication investigates the enzyme from Archaeoglobus fulgidus (Afu Pol-D). The C-terminal region of DP2 contains two conserved cysteine clusters, and their roles are investigated using site-directed mutagenesis. The cluster nearest the C terminus is essential for polymerase activity, and the cysteines are shown to serve as ligands for a single, critical Zn2 + ion. The cysteines farthest from the C terminal were not required for activity, and a role for these amino acids has yet to be defined. Additionally, it is shown that Afu Pol-D activity is slowed by the template strand hypoxanthine, extending previous results that demonstrated inhibition by uracil. Hypoxanthine was a weaker inhibitor than uracil. Investigations with isolated DP2, which has a measurable polymerase activity, localised the deaminated base binding site to this subunit. Uracil and hypoxanthine slowed Afu Pol-D “in trans”, that is, a copied DNA strand could be inhibited by a deaminated base in the alternate strand of a replication fork. The error rate of Afu Pol-D, measured in vitro, was 0.24 × 10− 5, typical for a polymerase that has been proposed to carry out genome replication in the Archaea. Deleting the 3′–5′ proofreading exonuclease activity reduced fidelity twofold. The results presented in this publication considerably increase our knowledge of Pol-D. Archaeal DNA Polymerase-D binds Zn, this is essential for activity. Archaeal DNA Polymerase-D is inhibited by uracil and hypoxanthine. Inhibition by deaminated bases occurs both in cis and in trans. Deaminated bases are sensed by the large (polymerase) subunit. Polymerase-D has high fidelity, compatible with genome replication.