A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type φ29 DNA polymerase
A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type φ29 DNA polymerase
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DOI：
10.1006/jmbi.1999.2900
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发表时间：
1999-07-02
影响因子：
5.6
通讯作者：
Salas, M
中科院分区：
文献类型：
--
作者：
Bonnin, A;Lázaro, JM;Salas, M
三个保守基序（称为 A、B 和 C）已被提议用于在所有类别的 DNA 依赖性聚合酶中形成聚合活性位点。在真核型（α 样）DNA 聚合酶中，基序 A 的特征在于共有“Dx(2)SLYP”。该保守基序的 phi 29 DNA 聚合酶残基 Tyr254 中的突变体先前已被证明在 dNTP 结合中受到影响。在这里，我们表明，Tyr254 单个取代为缬氨酸残基，使该酶能够掺入核糖核苷酸底物，而不影响其对 dNTP 的野生型亲和力。尽管野生型酶对 dNTP 的偏好程度是 rNTP 的两百万倍以上，但 Tyr254 突变为缬氨酸后，对 rNTP 的辨别能力降低了 1000 倍。除了这种基于糖选择的区分机制之外，phi 29 DNA 聚合酶在延伸 RNA 引物末端时效率非常低，导致其核酸外切降解。这些结果表明，phi 29 DNA 聚合酶的 Tyr254 负责区分传入核糖核苷酸的 2'-OH 基团。这是第一次，基序 A 的不变酪氨酸残基参与真核型 DNA 聚合酶中核糖核苷酸与脱氧核糖核苷酸的区分。 (C) 1999 年学术出版社。
Three conserved motifs (named A, B and C) have been proposed to form the polymerization active site in all classes of DNA-dependent polymerases. In eukaryotic-type (alpha-like) DNA polymerases, motif A is characterized by the consensus "Dx(2)SLYP". Mutants in phi 29 DNA polymerase residue Tyr254 of this conserved motif had been previously shown to be affected in dNTP binding. Here, we show that a single substitution of Tyr254 into a valine residue enables the enzyme to incorporate ribonucleotide substrates, without affecting its wild-type affinity for dNTPs. Whereas the wild-type enzyme preferred dNTPs more than two millionfold over rNTPs, the mutation of Tyr254 into valine reduced the discrimination for rNTPs up to 1000-fold. In addition to this discrimination mechanism, based on sugar selection, phi 29 DNA polymerase is very inefficient when extending an RNA primer terminus, allowing its exonucleolytic degradation. These results indicate that the Tyr254 of phi 29 DNA polymerase is responsible for the discrimination against the 2'-OH group of an incoming ribonucleotide. This is the first time that the invariant tyrosine residue of motif A is involved in ribo- versus deoxyribonucleotide discrimination in an eukaryotic-type DNA polymerase. (C) 1999 Academic Press.