Primer terminus stabilization at the 3'-5' exonuclease active site of phi 29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases
Primer terminus stabilization at the 3'-5' exonuclease active site of phi 29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases
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DOI：
10.1002/j.1460-2075.1996.tb00457.x
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发表时间：
1996-03-01
期刊：
影响因子：
11.4
通讯作者：
Blanco, L
中科院分区：
文献类型：
--
作者：
deVega, M;Lazaro, JM;Blanco, L
通过在pH29 DNA聚合酶中进行定点突变，我们分析了在大肠杆菌DNA聚合酶I中的两个进化保守的残基Thr358和Asn420的功能重要性，这两个残基属于依赖DNA聚合酶的3‘-5’外切酶结构域，结晶学分析表明它们直接作为3‘-5’外切酶活性部位的单链DNA(SsDNA)配体。在这些结构数据的基础上，Phi 29 DNA聚合酶Thr15和Asn62的相应残基的单一取代产生了结合单链DNA的能力非常降低或改变的酶，对这些突变衍生物在单链DNA底物上剩余的3‘-5’外切核酸酶活性的分析使我们能够得出结论，这两个残基在反应的催化中不起直接作用，另一方面，对匹配或错配的引物/模板结构上的3‘-5’外切核酸酶活性的分析表明，这两个高度保守的残基在聚合条件下的外切核酸解中起着关键作用，即在DNA聚合错误的校对中，这是大多数依赖DNA的DNA聚合酶的进化优势。此外，与先前观察到的作为金属配体的phi 29DNA聚合酶残基在3‘-5’外切核解和链置换中的双重作用相反，残基Thr15和Asn62的贡献似乎仅限于校对功能，这是通过稳定3‘-5’外切酶活性位点上磨损的引物末端来实现的。
By site-directed mutagenesis in phi 29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3'-5' exonuclease domain of DNA-dependent DNA polymerases, In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single-stranded DNA (ssDNA) ligands at the 3'-5' exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi 29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA, Analysis of the residual 3'-5' exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction, On the other hand, analysis of the 3'-5' exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i,e, in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA-dependent DNA polymerases. Moreover, in contrast to the dual role in 3'-5' exonucleolysis and strand displacement previously observed for phi 29 DNA polymerase residues acting as metal ligands, the contribution of residues Thr15 and Asn62 appears to be restricted to the proofreading function, by stabilization of the frayed primer-terminus at the 3'-5' exonuclease active site.