Reha-Krantz L J, Stocki S, Nonay R L, Dimayuga E, Goodrich L D, Konigsberg W H, Spicer E K
Department of Genetics, University of Alberta, Edmonton, Canada.
Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2417-21. doi: 10.1073/pnas.88.6.2417.
Classical genetic selection was combined with site-directed mutagenesis to study bacteriophage T4 DNA polymerase 3'----5' exonuclease activity. A mutant DNA polymerase with very little (less than or equal to 1%) 3'----5' exonuclease activity was generated. In vivo, the 3'----5' exonuclease-deficient DNA polymerase produced the highest level of spontaneous mutation observed in T4, 500- to 1800-fold above that of wild type. The large reduction in 3'----5' exonuclease activity appears to be due to two amino acid substitutions: Glu-191 to Ala and Asp-324 to Gly. Protein sequence similarities have been observed between sequences in the Escherichia coli DNA polymerase I 3'----5' exonuclease domain and conserved sequences in eukaryotic, viral, and phage DNA polymerases. It has been proposed that the conserved sequences contain metal ion binding ligands that are required for 3'----5' exonuclease activity; however, we find that some proposed T4 DNA polymerase metal binding residues are not essential for 3'----5' exonuclease activity. Thus, our T4 DNA polymerase studies do not support the hypothesis by Bernad et al. [Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219-228] that many DNA polymerases, including T4 DNA polymerase, share an extensively conserved 3'----5' exonuclease motif. Therefore, extrapolation from E. coli DNA polymerase I sequence and structure to other DNA polymerases for which there is no structural information may not be valid.
经典遗传选择与定点诱变相结合，以研究噬菌体T4 DNA聚合酶的3'→5'核酸外切酶活性。产生了一种3'→5'核酸外切酶活性极低（小于或等于1%）的突变DNA聚合酶。在体内，缺乏3'→5'核酸外切酶的DNA聚合酶产生了T4中观察到的最高自发突变水平，比野生型高500至1800倍。3'→5'核酸外切酶活性的大幅降低似乎是由于两个氨基酸取代：Glu-191突变为Ala以及Asp-324突变为Gly。已观察到大肠杆菌DNA聚合酶I的3'→5'核酸外切酶结构域中的序列与真核、病毒和噬菌体DNA聚合酶中的保守序列之间存在蛋白质序列相似性。有人提出，这些保守序列包含3'→5'核酸外切酶活性所需的金属离子结合配体；然而，我们发现一些提出的T4 DNA聚合酶金属结合残基对于3'→5'核酸外切酶活性并非必不可少。因此，我们对T4 DNA聚合酶的研究不支持Bernad等人[Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. & Salas, M. (1989) Cell 59, 219 - 228]提出的假设，即许多DNA聚合酶，包括T4 DNA聚合酶，共享一个广泛保守的3'→5'核酸外切酶基序。因此，从大肠杆菌DNA聚合酶I的序列和结构推断没有结构信息的其他DNA聚合酶可能是无效的。
