Morales J C, Kool E T
Department of Chemistry, University of Rochester, Rochester, New York 14627, USA.
Biochemistry. 2000 Oct 24;39(42):12979-88. doi: 10.1021/bi001578o.
Recent studies have identified amino acid side chains forming several hydrogen bonds in the DNA minor groove as potentially important in polymerase replication of DNA. Few studies have probed these interactions on the DNA itself. Using non-hydrogen-bonding nucleoside isosteres, we have now studied effects in both primer and template strands with several polymerases to investigate the general importance of these interactions. All six polymerases show differences in the H-bonding effects in the minor groove. Two broad classes of activity are seen, with a first group of DNA polymerases (KF(-), Taq, and HIV-RT) that efficiently extends nonpolar base pairs containing nucleoside Q (9-methyl-1H-imidazo[4,5-b]pyridine) but not the analogue Z (4-methylbenzimidazole), implicating a specific minor groove interaction at the first extension site. A second group of polymerases (Pol alpha, Pol beta, and T7(-)) fails to extend all non-H-bonding base pairs, indicating that these enzymes may need minor groove hydrogen bonds at both minor groove sites or that they are especially sensitive to noncanonical DNA structure or stability. All DNA polymerases examined use energetically important minor groove interactions to probe newly synthesized base pairs before extending them. The positions of these interactions vary among the enzymes, and only a subset of the interactions identified structurally appears to be functionally important. In addition, polymerases appear to be differently sensitive to small changes in base pair geometry.
最近的研究已确定，在DNA小沟中形成多个氢键的氨基酸侧链在DNA聚合酶复制过程中可能具有重要作用。很少有研究在DNA自身上探究这些相互作用。利用非氢键核苷类似物，我们现在用几种聚合酶研究了引物链和模板链中的效应，以调查这些相互作用的普遍重要性。所有六种聚合酶在小沟中的氢键效应都存在差异。观察到两种广泛的活性类型，第一组DNA聚合酶（KF(-)、Taq和HIV-RT）能有效延伸含核苷Q（9-甲基-1H-咪唑并[4,5-b]吡啶）的非极性碱基对，但不能延伸类似物Z（4-甲基苯并咪唑），这表明在第一个延伸位点存在特定的小沟相互作用。第二组聚合酶（Polα、Polβ和T7(-)）不能延伸所有非氢键碱基对，这表明这些酶可能在两个小沟位点都需要小沟氢键，或者它们对非经典DNA结构或稳定性特别敏感。所有检测的DNA聚合酶在延伸新合成的碱基对之前，都会利用能量上重要的小沟相互作用来探测它们。这些相互作用的位置在不同酶之间有所不同，并且在结构上确定的相互作用中只有一部分似乎在功能上是重要的。此外，聚合酶对碱基对几何形状的微小变化似乎有不同的敏感性。
