DNA Replication Fidelity

工作总结：该项目的长期目标是通过多蛋白DNA复制和修复复合物了解DNA合成的保真度。今年，在三个领域取得了进展。我们在酵母细胞中建立了一个体内系统，以确定由前导链和滞后链DNA复制机制的DNA复制的保真度，并使用该系统来证明酵母起源建立由两个碱基类似物诱导的复制诱变的链偏差，其中之一（8-氧代鸟嘌呤）是由氧化应激产生的主要DNA损伤。我们研究了酵母中的translesion合成聚合酶在暴露于模拟阳光下的作用。这些研究与这些聚合酶在抑制或可能促进皮肤癌中的真实生理作用比所有先前的由254 nm UV光照射诱导的细胞毒性和诱变的研究更相关，因为由阳光产生的损伤的光谱显著不同（例如，更多的氧化应激）。今年，我们还描述了一种测定方法来测量用纯化酶重建的BER的保真度，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。发现Pol β依赖性BER的碱基取代保真度比单独由Pol β进行的单核苷酸空位填充高3至8倍，这表明BER反应中的其他蛋白质可以在单核苷酸BER期间增强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