Structure-Function Studies Of DNA Replication Fidelity

工作总结：该项目的长期目标是（1）了解DNA复制和修复过程的保真度和效率，这些过程控制从病毒到人类的生物体中的细胞存活和基因组稳定性，以及（2）了解环境压力如何干扰这些过程，导致细胞毒性，诱变和对人类健康的不利影响。今年，在理解几种不同的DNA聚合酶的功能方面取得了进展，包括复制型T7 DNA聚合酶（A家族）、核复制型pols α和δ（B家族）、DNA修复pols λ和mu（X家族）、跨损伤合成pols zeta（B家族）和pols eta、kappa和iota（Y家族）。这些研究揭示了细胞准确复制正常DNA以及被环境因子破坏的DNA所使用的策略，以避免诱变和细胞毒性以及伴随的不良健康后果。本项目已与Z 01-ES-65046合并。 在复制之前，有几个修复过程起作用，以去除内源性细胞代谢或暴露于环境应激所产生的DNA损伤。这些修复过程为高效和高度准确的DNA复制提供了未受损的底物。高复制保真度取决于DNA聚合酶选择正确而不是不正确的核苷酸掺入DNA而不添加或删除核苷酸的能力。这种选择性是复制叉处以及与修复相关的DNA合成期间保真度的主要决定因素。错配的核酸外切酶校正可以进一步增加DNA复制保真度。当DNA损伤在复制之前没有修复时，某些损伤可以阻止复制叉的进展。在这些情况下，细胞周期检查点、跨损伤合成反应和重组修复过程起作用以维持基因组稳定性并改善细胞存活。本项目通过研究A、B、X、Y和RT家族DNA聚合酶及其辅助蛋白的结构、生化性质和体内推定功能之间的关系来研究这些复制保真度过程。采用双重方法，包括体外生物化学研究和酵母、小鼠和人类的体内遗传研究。

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 replicative T7 DNA polymerase (family A), nuclear replicative pols alpha and delta (family B), DNA repair pols lambda and mu (family X), and translesion synthesis pol zeta (family B) and pols eta, kappa and 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.