Bonnin A, Lázaro J M, Blanco L, Salas M
Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, 28049, Spain.
J Mol Biol. 1999 Jul 2;290(1):241-51. doi: 10.1006/jmbi.1999.2900.
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 "Dx2SLYP". Mutants in phi29 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 million-fold 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, phi29 DNA polymerase is very inefficient when extending an RNA primer terminus, allowing its exonucleolytic degradation. These results indicate that the Tyr254 of phi29 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.
已经提出在所有类型的依赖DNA的聚合酶中，三个保守基序（命名为A、B和C）形成聚合活性位点。在真核生物类型（α样）的DNA聚合酶中，基序A的特征是共有序列“Dx2SLYP”。此前已表明，φ29 DNA聚合酶中该保守基序的残基Tyr254的突变体在dNTP结合方面受到影响。在这里，我们表明，将Tyr254单个替换为缬氨酸残基可使该酶掺入核糖核苷酸底物，而不影响其对dNTP的野生型亲和力。野生型酶对dNTP的偏好比对rNTP高两百多万倍，而将Tyr254突变为缬氨酸会使对rNTP的辨别能力降低至1000倍。除了这种基于糖选择的辨别机制外，φ29 DNA聚合酶在延伸RNA引物末端时效率非常低，会使其发生核酸外切降解。这些结果表明，φ29 DNA聚合酶的Tyr254负责辨别进入的核糖核苷酸的2'-OH基团。这是基序A中不变的酪氨酸残基首次参与真核生物类型DNA聚合酶中核糖核苷酸与脱氧核糖核苷酸的辨别。
