Structure-Function Studies Of DNA Replication Fidelity

工作总结：该项目的长期目标是(1)了解从病毒到人类的生物体中控制细胞生存和基因组稳定的DNA复制和修复过程的保真度和效率，以及(2)了解环境应激如何扰乱这些过程，导致细胞毒性、突变和对人类健康的不利影响。今年，在了解几种不同的DNA聚合酶的功能方面取得了进展，包括线粒体PolGamma和T7DNA聚合酶(A家族)、核复制PolDelta(B家族)、DNA修复极lambda和Mu(X家族)和跨损伤合成Poliota(Y家族)。这些研究揭示了细胞用来准确复制正常DNA以及被环境因素破坏的DNA的策略，以避免突变和细胞毒性以及随之而来的不利健康后果。该项目已与Z01-ES-65046相结合。 有几个修复过程在复制之前进行，以消除内源性细胞新陈代谢或暴露在环境压力下造成的DNA损伤。这些修复过程为高效和高精度的DNA复制提供了未受损的底物。高复制保真度取决于DNA聚合酶在不增加或删除核苷酸的情况下选择正确的核苷酸而不是错误的核苷酸并入DNA的能力。这种选择性是复制分叉以及与修复相关的DNA合成过程中保真度的主要决定因素。对错配的核外溶解校对可以进一步提高DNA复制的保真度。当DNA损伤在复制之前没有修复时，某些损伤可能会阻碍复制分叉的进展。在这种情况下，细胞周期检查点、跨损伤合成反应和重组修复过程运行，以维持基因组稳定和提高细胞存活率。本项目通过研究A、B、X、Y和RT家族DNA聚合酶的结构与它们的辅助蛋白之间的关系、它们的生化特性以及它们在体内的可能功能来研究这些复制保真过程。使用双重方法，包括体外生化研究和体内遗传研究，在酵母、小鼠和人类中进行。

Summary of Work: The long-term goals of this project are (1) to understand the fidelity and efficiency of the DNA replication and repair processes that control cell survival and genome stability in organisms from viruses to humans, and (2) to understand how environmental stress perturbs these processes, resulting in cytotoxicity, mutagenesis and adverse effects on human health. This year, progress was made towards understanding the functions of several different DNA polymerases, including mitochondrial pol gamma and T7 DNA polymerase (family A), nuclear replicative pol delta (family B), DNA repair pols lambda and mu (family X), and translesion synthesis pol iota (family Y). These studies are revealing the strategies used by cells to accurately replicate normal DNA as well as DNA damaged by environmental agents, in a manner that avoids mutagenesis and cytotoxicity and the accompanying adverse health consequences. This project has been combined with Z01-ES-65046. Several repair processes operate prior to replication to remove DNA damage generated by endogenous cellular metabolism or exposure to environmental stress. These repair processes provide undamaged substrates for efficient and highly accurate DNA replication. High replication fidelity depends on the ability of DNA polymerases to select correct rather than incorrect nucleotides for incorporation into DNA without adding or deleting nucleotides. This selectivity is a prime determinant of fidelity at the replication fork and also during DNA synthesis associated with repair. Exonucleolytic proofreading of mismatches can further increase DNA replication fidelity. When DNA damage is not repaired prior to replication, certain lesions can stall progression of the replication fork. Under these circumstances, cell cycle checkpoints, translesion synthesis reactions and recombinational repair processes operate to maintain genome stability and improve cell survival. This project investigates these replication fidelity processes by studying the relationships between the structures of A, B, X, Y and RT family DNA polymerases and their accessory proteins, their biochemical properties and their putative functions in vivo. A dual approach is used involving in vitro biochemical studies with in vivo genetic studies in yeast and mice and humans.