Mechanisms of damaged DNA replication in eukaryotes

描述(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损伤会阻止经典DNA聚合酶的复制。因此，细胞拥有多种非经典的DNA聚合酶，可以取代停滞在DNA损伤部位的经典聚合酶，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不同以及它们如何适应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如何被招募到DNA损伤部位，如何将停滞不前的经典聚合酶从DNA损伤部位取代，以及复制辅助因子如何促进核苷酸掺入，以对抗非经典聚合酶对DNA的损伤。为了解决这些问题，我们提出了以下三个具体目标的研究：(1)确定其他蛋白质因素对非经典聚合酶对DNA损伤的核苷酸掺入的影响；(2)确定跨损伤合成过程中非经典聚合酶重新聚集的机制；(3)确定跨损伤合成过程中经典聚合酶置换的机制。这些研究将使人们清楚地了解非经典聚合酶如何在DNA损伤部位取代经典聚合酶，以及其他蛋白质因素如何促进DNA损伤的复制。此外，这些研究将有助于我们了解突变和癌症的起源，并将为它们的预防提供洞察力。

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.