Mechanisms of damaged DNA replication in eukaryotes

描述（由申请人提供）：我们研究的长期目标是在热力学，动力学和结构水平上了解真核生物中受损DNA的复制。模板链中的DNA损伤阻断了经典DNA聚合酶的复制。因此，细胞具有多种非经典DNA聚合酶，这些非经典DNA聚合酶可以取代DNA损伤位点的经典聚合酶，并可以通过损伤进行复制。最近的动力学研究和结构研究为这些非经典聚合酶与经典聚合酶的区别以及它们如何能够适应DNA损伤提供了实质性的见解。然而，目前尚不清楚非经典聚合酶是如何被招募到DNA损伤位点的，停滞的经典聚合酶是如何从DNA损伤位点转移的，以及复制辅助因子是如何促进非经典聚合酶在DNA损伤的相反方向整合核苷酸的。为了解决这些问题，我们提出了以下三个具体的研究目标：(1)确定其他蛋白质因子对非经典聚合酶对核苷酸结合的影响；(2)确定翻译合成过程中非经典聚合酶募集的机制；(3)确定翻译合成过程中经典聚合酶置换的机制。这些研究将使我们更清楚地了解非经典聚合酶是如何在DNA损伤位点取代经典聚合酶的，以及其他蛋白质因子如何促进受损DNA的复制。此外，这些研究将有助于我们了解突变和癌症的起源，并将为预防它们提供见解。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the replication of damaged DNA in eukaryotes at the thermodynamic, kinetic, and structural levels. DNA damage in the template strand blocks replication by classical DNA polymerases. Consequently cells possess a variety of non-classical DNA polymerases that can replace the classical polymerase stalled at sites of DNA damage and can replicate through the damage. Recent kinetic studies and structural studies have provided substantial insights into how these non-classical polymerases differ from classical polymerases and how they are able to accommodate DNA damage. It remains unclear, however, how non-classical polymerases are recruited to sites of DNA damage, how stalled classical polymerases are displaced from sites of DNA damage, and how replication accessory factors promote nucleotide incorporation opposite DNA damage by non-classical polymerases. To address these issues, we propose studies with the following three specific aims: (1) to determine the effect of other protein factors on nucleotide incorporation opposite DNA damage by non-classical polymerases, (2) to determine the mechanism of non-classical polymerase recruitment during translesion synthesis, and (3) to determine the mechanism of classical polymerase displacement during translesion synthesis. These studies will provide a clear understanding of exactly how non-classical polymerases replace classical polymerases at sites of DNA damage and how other protein factors contribute to the replication of damaged DNA. Furthermore, these studies will contribute to our understanding of the origins of mutations and cancers and will provide insight into their prevention.