PROBING STRUCTURE-FUNCTION RELATIONSHIPS WITH DNA POLYMERASES

我们正在使用通过重组DNA技术获得的突变酶， 研究结构和动力学性质之间的关系 DNA聚合酶和它们的保真度。 重点是聚合酶， 其中X射线晶体结构信息是或可能很快成为 available. 我们已经建立了持续性和错误特异性 在两种反应pH下的核酸外切酶缺陷型Klenow聚合酶。 我们 将分析扩展到三种突变体，其中酪氨酸残基在 在聚合酶活性位点的“O螺旋”中的766位发生改变 连接到丙氨酸、丝氨酸或苯丙氨酸，以及连接到另外的活性位点 参与催化和金属结合的残基。 几个这样 突变体改变了持续合成能力和保真度，有些突变体 mutator和其他antimutator表型。 数据表明，dNTP 和金属结合残基对于确定碱基 选择性，但（迄今为止）不是模板-引物错配引发的 错误. 我们目前正在研究HIV-1的突变衍生物， 逆转录酶，其中我们突变了“拇指”残基， 已知对于结合双链模板-引物是重要。 我们 也开始建立野生型T4和T7 DNA的保真度 聚合酶，以及它们的核酸外切酶缺陷的合作伙伴不同， 一个和两个保守氨基酸。 重组分析 DNA聚合酶，其相当多的遗传和/或动力学数据是已知的。 这将提高我们对精确DNA合成的理解， DNA加合物是如何影响保真度的

We are using mutant enzymes obtained by recombinant DNA technology to examine the relationship between the structural and kinetic properties of DNA polymerases and their fidelity. Emphasis is on polymerases for which X-ray crystal structure information is or may soon become available. We have established the processivity and error specificity of exonuclease-deficient Klenow polymerase at two reaction pHs. We extended the anlaysis to three mutants in which a tyrosine residue at position 766, in the "O helix" of the polymerase active site, was changed to an alanine, serine or phenylalanine, and to additional active site residues involved in catalysis and metal binding. Several of these mutants have altered processivity and altered fidelity, some having mutator and others antimutator phenotypes. The data suggest that dNTP and metal binding residues are important for determining base selectivity, but (to date) not template-primer misalignment-initiated errors. We are presently examining mutant derivatives of the HIV-1 reverse transcriptase, in which we have mutated the "thumb" residues known to be important for binding double-stranded template-primer. We have also begun to establish the fidelity of the wild-type T4 and T7 DNA polymerase, as well as their exonuclease-deficient partners differing by one and two conserved amino acids, respectively. Analyses of recombinant DNA polymerases for which considerable genetic and/or kinetic data are available should improve our understanding of accurate DNA synthesis and how fidelity is affected by DNA adducts.