Use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA polymerases.
Use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA polymerases.
复制标题
使用 2-氨基嘌呤荧光研究 β 发夹在噬菌体 T4 和 RB69 DNA 聚合酶催化的校对途径中的作用。
DOI：
10.1021/bi800211f
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Reha-Krantz,LindaJ
中科院分区：
文献类型：
--
作者：
Subuddhi,Usharani;Hogg,Matthew;Reha-Krantz,LindaJ
为了使DNA聚合酶校正错误掺入的核苷酸，引物链的末端3 - 4个核苷酸必须在结合到核酸外切酶活性中心之前与模板链分离。噬菌体T4 DNA聚合酶的遗传和生物化学研究表明，需要核酸外切酶结构域中突出的β-发夹结构来有效地形成链分离的核酸外切酶复合物。我们在这里提出进一步的突变分析的T4 DNA聚合酶β-发夹结构的环区，这提供了额外的证据，在环中的残基，即Y254和G255，是重要的DNA复制保真度。利用碱基类似物2-氨基嘌呤（2AP）和突变型RB 69 DNA聚合酶的荧光在体外反应中探索了链分离的机制，突变型RB 69 DNA聚合酶对β发夹、核酸外切酶活性位点或两者都进行了修饰。我们从这些研究中提出，在T4和RB 69 DNA聚合酶的核酸外切酶结构域中的β发夹的功能，以促进链分离，但在核酸外切酶活性中心的残基需要捕获的3′端的引物链后链分离。
For DNA polymerases to proofread a misincorporated nucleotide, the terminal 3−4 nucleotides of the primer strand must be separated from the template strand before being bound in the exonuclease active center. Genetic and biochemical studies of the bacteriophage T4 DNA polymerase revealed that a prominent β-hairpin structure in the exonuclease domain is needed to efficiently form the strand-separated exonuclease complexes. We present here further mutational analysis of the loop region of the T4 DNA polymerase β-hairpin structure, which provides additional evidence that residues in the loop, namely, Y254 and G255, are important for DNA replication fidelity. The mechanism of strand separation was probed inin vitroreactions using the fluorescence of the base analogue 2-aminopurine (2AP) and mutant RB69 DNA polymerases that have modifications to the β hairpin, to the exonuclease active site, or to both. We propose from these studies that the β hairpin in the exonuclease domain of the T4 and RB69 DNA polymerases functions to facilitate strand separation, but residues in the exonuclease active center are required to capture the 3′ end of the primer strand following strand separation.