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， 技术来研究结构和动力学之间的关系， DNA聚合酶的性质及其持续合成能力和保真度。 重点是酶的结构信息是可用的， 包括Klenow聚合酶和DNA聚合酶β。我们已经确定 外切核酸酶缺陷型Klenow细胞持续合成能力和错误特异性 聚合酶和22个突变衍生物在几个关键参数中改变。 这些突变体中有几个已经改变了持续合成能力。两人强烈 降低的保真度，其中之一选择性地增强了 嘧啶dNTPs。另一个则大大提高了忠诚度。 我们现在 确定这是否反映了在dNTP期间增强的辨别力 插入和/或错配延伸或在结合到配对或 错配的模板引物。Klenow数据表明，dNTP和金属 结合残基对于确定取代保真度是重要的，但 而不是模板引物错误引发的错误。我们还 鉴定了一种Klenow移码突变聚合酶， 聚合酶中氨基酸缺失导致的持续合成能力 “拇指”域。我们已经确定了对保真度的影响， 聚合酶辅助蛋白，增强持续合成能力，证明 在控制移码忠实度方面有很强的作用。 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，以检查核酸外切 简单重复序列复制过程中的校对和错误率。 本研究表明，校对对语码转换的贡献 均聚物运行中的保真度随着运行长度的增加而降低。的 研究现在扩展到二核苷酸和三核苷酸重复， 在癌细胞和几种遗传性疾病中不稳定。我们有 启动了β聚合酶突变形式的研究， 三元酶结构信息DNAdNTP复合物。 目前的重点是氨基酸残基假设，使 接触对于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提高我们的 了解人类基因组如何稳定复制， 维持，以及DNA加合物如何影响Gemone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.