Non-template-directed nucleotide addition by human DNA polymerases

描述（由申请人提供）：本项目旨在了解人类DNA聚合酶在DNA末端非模板定向核苷酸添加的分子机制。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的基本问题。非模板定向核苷酸添加是一些DNA聚合酶合成DNA的能力，尽管没有模板来指导特定核苷酸的优先插入。这可能发生在DNA末端，特别是由破坏剂（如电离辐射）或复制叉崩溃产生的末端。在人类细胞中，DNA聚合酶如何与链断裂相互作用并合成DNA尚不完全清楚。为了解决这个问题，我们设计了一组实验来测试来自三个不同结构家族的人类DNA聚合酶是否可以进行非模板添加。这里描述的项目使用体外方法来检查DNA末端的哪些结构特征对于每个聚合酶的非模板添加至关重要。此外，还提出了实验来检查在复制和修复中重要的其他蛋白质是否需要非模板添加。目标是更全面地了解核苷酸是如何通过DNA聚合酶添加到生长中的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，它将有助于我们理解诱变的分子基础以及癌症和衰老等生物过程。作为一个比较模型，它也将提高我们对核苷酸正常DNA合成工作的理解。这项提案有很强的教育成分，因为它旨在鼓励本科生参与，特别是那些传统上在学术研究中代表性不足的群体。

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.