Fidelity of DNA Synthesis

描述（由申请人提供）：DNA聚合酶是至关重要的酶，目前尚未充分探索癌症化疗的靶点。更好地了解单个真核DNA聚合酶在DNA合成过程中的功能及其催化机制，对于基本的生物学理解和癌症化疗方案的潜在开发都具有重要意义。我们建议在这里的真核生物DNA聚合酶的调查，利用我们的能力，以创建具有不同的催化性能的突变DNA聚合酶的大型库。在具体目标1中，我们将鉴定DNA聚合酶-d、-e和-a的突变体，这些突变体包含特定的诱变核苷酸类似物。我们将通过用相应的突变酶替换内源性聚合酶来分析这些聚合酶在DNA代谢中的作用。在经历复制、修复或重组的细胞中掺入类似物和/或诱导诊断突变将为每种聚合酶参与这些过程提供标记。在特定目标2中，我们将利用我们的新系统进行体内蛋白质进化，以创建新的DNA聚合酶-6突变体，其具有增强的复制含有大体积病变的DNA的能力，并且我们将分析增加病变旁路对细胞存活和诱变的后果。在具体目标3中，我们将通过创建和研究增变子和反增变子突变体来分析DNA聚合酶选择互补核苷酸底物的机制。拟议的研究应有助于开发特定的DNA聚合酶作为化疗靶点，通过提供相关的知识，抑制其催化活性和/或破坏其在癌细胞中的功能。

DESCRIPTION (provided by applicant): DNA polymerases are critically important enzymes and present targets for cancer chemotherapy that have not been adequately explored. Greater knowledge of the functions of individual eukaryotic DNA polymerases in DNA synthetic processes, and of their catalytic mechanisms, is significant for both fundamental biologic understanding and potential exploitation in cancer chemotherapeutic regimens. We propose here an investigation of eukaryotic DNA polymerases that takes advantage of our ability to create large libraries of mutant DNA polymerases with different catalytic properties. In Specific Aim 1, we will identify mutants of DNA polymerases-d, -e, and -a that incorporate specific, mutagenic nucleotide analogs. We will analyze the roles of these polymerases in DNA metabolism by replacing the endogenous polymerases with the corresponding mutant enzymes. Incorporation of the analog and/or induction of diagnostic mutations in cells undergoing replication, repair or recombination will provide a signature for the participation of each polymerase in these processes. In Specific Aim 2, we will utilize our new system for in vivo protein evolution to create novel DNA polymerase-6 mutants with enhanced capacity to copy DNA containing bulky lesions, and we will analyze the consequences of increased lesion bypass for cell survival and mutagenesis. In Specific Aim 3, we will analyze the mechanisms by which DNA polymerases select complementary nucleotide substrates by creating and studying mutator and anti-mutator mutants. The proposed studies should facilitate exploitation of specific DNA polymerases as chemotherapeutic targets by providing knowledge relevant to inhibiting their catalytic activities and/or disrupting their functions in cancer cells.