PROBING STRUCTURE FUNCTION RELATIONSHIPS WITH DNA POLYMERASES

我们正在使用通过重组DNA获得的突变DNA聚合酶 技术来考察结构和动力学之间的关系 DNA聚合酶的性质及其加工性和保真度。 重点放在结构信息可用的酶上， 包括Klenow聚合酶和DNA聚合酶β。我们已经决定 核酸外切酶缺陷Klenow的过程性和错误特异性 聚合酶和22个突变的衍生物在几个关键参数上发生了变化。 这些突变体中有几个已经改变了加工能力。有两个人强烈地 降低保真度，其中之一选择性地增强了 嘧啶脱氧核糖核酸。另一种则大大提高了保真度。我们现在是 确定这是否反映了dNTP期间歧视的增强 插入和/或错配延伸或在绑定到配对或 错配的模板-引物。Klenow的数据表明dNTP和Metals 结合残基对于确定取代的保真度很重要，但是 不是模板-引物未对齐-引发的错误。我们还有 鉴定一个Klenow移码突变聚合酶 由于聚合酶中氨基酸缺失而导致的加工性 “拇指”域。我们已经确定了对保真度的影响 增强加工性的聚合酶辅助蛋白，演示 在控制帧转换保真度方面发挥强大的作用。我们已经建立了 几种野生型和核酸外切酶缺陷型的保真度 聚合酶与核外溶解的关系 简单重复序列复制过程中的校对和错误率。 本研究表明，校对对移码的贡献 均聚游程中的保真度随着游程长度的增加而降低。这个 研究现在正在扩展到二核苷酸和三核苷酸重复序列， 在癌细胞和一些遗传性疾病中是不稳定的。我们有 启动了对β聚合酶突变形式的研究，基于唯一的 三元酶DNAdNTP络合物的结构信息。 目前重点放在氨基酸残基上，假设 接触对核苷酸选择性很重要。我们相信 DNA聚合酶的结构功能研究将改善我们的 了解人类基因组是如何稳定复制的 以及DNA加合物如何影响配子体的稳定性。

We are using mutant DNA polymerases obtained by recombinant DNA technology to examine the relationship between the structural and kinetic properties of DNA polymerases and their processivity and fidelity. Emphasis is on enzymes for which structural information is available, including Klenow polymerase and DNA polymerase beta. We have determined the processivity and error specificity of exonuclease-deficient Klenow polymerase and 22 mutant derivatives altered in several key parameters. Several of these mutants have altered processivity. Two have strongly reduced fidelity, one of which selectively enhances misincorporation of pyrimidine dNTPs. Another has strongly increased fidelity. We are now determining if this reflects enhanced discrimination during dNTP insertion and/or mispair extension or during binding to paired or mispaired template-primers. The Klenow data suggest that dNTP and metal binding residues are important for determining substitution fidelity, but not template-primer misalignment-initiated errors. We have also identified a Klenow frameshift mutator polymerase with reduced processivity that results from deletion of amino acids in the polymerase "thumb" domain. We have determined the effects on fidelity of a polymerase accessory protein that enhances processivity, demonstrating a strong role in controlling frameshift fidelity. We have establish the fidelity of the wild-type and exonuclease-deficient forms of several polymerases to examine the relationship between exonucleolytic proofreading and error rates during copying of simple repeat sequences. This study shows that the contribution of proofreading to frameshift fidelity in homopolymeric runs decreases as the run length increases. The study is now being extended to the di- and tri-nucleotide repeats that are unstable in cancer cells and several hereditary diseases. We have initiated a study of mutant forms of beta polymerase based on the only structural information available for a ternary enzymeDNAdNTP complex. Emphasis is currently on amino acid residues hypothesized to make contacts important for nucleotide selectivity. It is our belief that structure function studies of DNA polymerases will improve our understanding of how the human genome is stably replicated and maintained, and how DNA adducts affect gemone stability.