Non-template-directed nucleotide addition by human DNA polymerases

人类 DNA 聚合酶的非模板定向核苷酸添加

• 批准号: 7126975
• 负责人: ANGEL L ISLAS
• 金额: $ 20.2万
• 依托单位: SANTA CLARA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7126975
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA repair; DNA replication; catalyst; chemical addition; chemical kinetics; electrophoresis; enzyme activity; enzyme mechanism; intermolecular interaction; nucleotide metabolism

DESCRIPTION (provided by applicant): This project seeks to understand the molecular mechanism of non-template-directed nucleotide addition by human DNA polymerases at DNA termini. It therefore seeks to understand the basic problem of errors in nucleotide selection by DNA polymerases from a novel perspective. Non-template-directed nucleotide addition is the ability of some DNA polymerases to synthesize DNA in spite of the absence of a template to direct the preferential insertion of a particular nucleotide. This can occur at DNA ends, especially ends created by either damaging agents (e.g. ionizing radiation) or replication fork collapse. How DNA polymerases interact with strand breaks and synthesize DNA in human cells is not fully known. To address this problem we have designed a set of experiments to test whether human DNA polymerases from three different structural families of polymerases can perform non-templated addition. The projects described here use an in vitro approach to examine what structural features of DNA ends are critical for non-templated addition in each polymerase. Also, experiments are proposed to examine whether other proteins, important in replication and repair, are necessary for non-templated addition. The goal is to have a more complete understanding of how nuceleotides are added to growing DNA molecules by DNA polymerases. The significance of understanding how human DNA polymerases add nucleotides without template direction rests in the insights it will shed on our understanding of molecular basis of mutagenesis and biological processes like cancer and aging. As a comparative model, it will also improve our understanding of nucleotide normal DNA synthesis works. This proposal has a strong educational component in that is seeks to encourage undergraduate participation, especially from those groups who have been traditionally under- represented in academic research.

描述(申请人提供)：这个项目试图了解人类DNA聚合酶在DNA末端进行非模板导向的核苷酸加成的分子机制。因此，它试图从一个新的角度来理解DNA聚合酶在核苷酸选择中的错误这一基本问题。非模板导向的核苷酸加成是一些DNA聚合酶在没有模板的情况下合成DNA的能力，它可以指导特定核苷酸的优先插入。这可能发生在DNA末端，特别是由损害剂(例如电离辐射)或复制叉崩溃造成的末端。DNA聚合酶如何与人类细胞中的链断裂和合成DNA相互作用尚不完全清楚。为了解决这个问题，我们设计了一系列实验来测试来自三个不同聚合酶结构家族的人DNA聚合酶是否可以进行非模板加成。这里描述的项目使用体外方法来检查DNA末端的什么结构特征对于每个聚合酶中的非模板添加是关键的。此外，还建议进行实验，以检查在复制和修复中重要的其他蛋白质是否对非模板添加是必要的。其目标是更全面地了解DNA聚合酶是如何将核苷酸添加到不断增长的DNA分子中的。理解人类DNA聚合酶如何在没有模板方向的情况下添加核苷酸的意义在于它将基于我们对突变的分子基础以及癌症和衰老等生物过程的理解。作为一个比较模型，它也将提高我们对核苷酸正常DNA合成工作原理的理解。这项建议具有很强的教育成分，它旨在鼓励本科生的参与，特别是那些传统上在学术研究中代表性不足的群体的参与。