Unique error signature of the four-subunit yeast DNA polymerase ε
Unique error signature of the four-subunit yeast DNA polymerase ε
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DOI：
10.1074/jbc.m306893200
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发表时间：
2003-10-31
影响因子：
4.8
通讯作者：
Kunkel, TA
中科院分区：
文献类型：
--
作者：
Shcherbakova, PV;Pavlov, YI;Kunkel, TA
我们从酿酒酵母中纯化了野生型和核酸外切酶缺陷型四亚基DNA聚合酶复合物，并分析了这两种酶合成DNA的保真度。野生型Pol突变体准确地合成DNA，分别以小于或等于2 × 10（-5）和小于或等于5 × 10（-7）的平均错误率产生单碱基取代和缺失。缺乏3' -> 5'外切核酸酶活性的Pol β在一定程度上较不准确，这表明野生型Pol β校正了聚合酶产生的至少92%的碱基取代错误和至少99%的移码错误。令人惊讶的是，核酸外切酶缺陷型聚合酶的碱基取代保真度比其他复制型聚合酶的校对缺陷型低几倍。此外，错误谱显示了在其他A、B、C或X家族聚合酶中未观察到的特征：由T引起的高比例的颠换。dTTP、T . dCTP和C . dTTP错配。这种独特的错误特异性和氨基酸序列比对表明，聚合酶活性位点的结构的Pol？B不同于其他B家族成员。我们观察到的相似性和差异的频谱所产生的校正缺陷的Pol的校正和DNA错配修复缺陷的酵母菌株在体外和体内发生的取代。我们讨论了这些研究结果的影响，在DNA复制中的聚合酶活性的作用。
We have purified wild type and exonuclease-deficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol epsilon synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of less than or equal to 2 x 10(-5) and less than or equal to 5 x 10(-7), respectively. Pol epsilon lacking 3' --> 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T . dTTP, T . dCTP, and C . dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol epsilon differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol epsilon polymerase activity in DNA replication.