DNA Replication Fidelity

工作概述：该项目的长期目标是了解多蛋白DNA复制和修复复合物合成DNA的保真度。今年，我们在三个方面取得了进展。我们在酵母细胞中建立了一个体内系统，通过前链和后链DNA复制机制来确定DNA复制的保真度，并利用该系统证明酵母起源建立了由两个碱基类似物诱导的复制诱变的链偏倚，其中一个（8-氧鸟嘌呤）是氧化应激产生的主要DNA损伤。我们研究了翻译合成聚合酶在酵母暴露于模拟阳光下的作用。这些研究更关注这些聚合酶在抑制或可能促进皮肤癌方面的真实生理作用，而不是之前所有关于254 nm紫外光照射诱导的细胞毒性和诱变的研究，因为阳光产生的病变光谱明显不同（例如，更多的氧化应激）。今年，我们还描述了一种测定用纯化酶重建的BER保真度的方法，并用它来确定涉及Pol β的四蛋白单核苷酸BER反应的保真度。Pol β依赖的BER的碱基取代保真度比Pol β单独填补的单核苷酸间隙高3 ~ 8倍，这表明在单核苷酸BER反应中，其他蛋白质可能会提高Pol β的碱基取代保真度

Summary of Work: The long-term goal of this project is to understand the fidelity of DNA synthesis by multiprotein DNA replication and repair complexes. This year, progress was made in three areas. We established an in vivo system in yeast cells to determine the fidelity of DNA replication by the leading and lagging strand DNA replication machinery and used the system to demonstrate that yeast origins establish a strand bias for replicational mutagenesis induced by two base analogs, one of which (8-oxo-guanine) is a major DNA lesion generated by oxidative stress. We investigated the roles of translesion synthesis polymerases in yeast upon exposure to simulated sunlight. These studies are more relevant to the true physiological roles of these polymerases in suppressing or perhaps promoting skin cancer than are all previous studies of cytotoxicity and mutagenesis induced by irradiation with 254 nm UV light, because the spectrum of lesions generated by sunlight are significantly different (e.g., much more oxidative stress). This year, we also described an assay to measure the fidelity of BER reconstituted with purified enzymes, and used it to determine the fidelity of a four-protein single-nucleotide BER reaction involving Pol beta. The base substitution fidelity of Pol beta dependent BER was found to be 3 to 8-fold higher than is single nucleotide gap filling by Pol beta alone, suggesting that other proteins in the BER reaction may enhance the base substitution fidelity of Pol beta during single-nucleotide BER