ACCURACY OF DNA REPLICATION IN VITRO

这个项目的长期目标是确定DNA的精细结构 复制保真度。人类基因组的复制涉及到许多 复杂的反应，包括在起源时启动合成， 超前股和滞后股的延长和更换 带有DNA的RNA引物。目前的模型表明，不止一个DNA 聚合酶是复制所必需的，即启动或 精加工链可能与执行大部分链的链不同 伸长，以及复制领先和滞后的蛋白质 股可能是不同的。以确定这些差异是否会导致 在不同的复制错误率下，我们正在检查 真核DNA聚合酶催化的DNA合成 Epsilon和Gamma与双向DNA复制的保真度 人HeLa细胞提取物中的多蛋白复制装置。 我们发现DNA聚合酶有明显不同的误差 利率和特殊性，这对他们在 DNA复制的不同阶段。此外，尽管整体保真度 在两条链上复制的情况相似，碱基替换和 帧移位错误率在某些站点上确实不同 落后的链复制设备。最后，为了更好地 了解已知的诱变剂和致癌物对保真度的影响 关于DNA合成，我们正在进行复制保真度的研究 含有明确损伤的DNA分子，包括补骨脂素单加合物， 紫外线感光产品和AAF加合物。我们打算继续这些研究 了解每个聚合反应的保真度 人类基因组的完全复制所需的。

The long-term goal of this project is to define the fine structure of DNA replication fidelity. Replication of the human genome involves a number of complex reactions including initiation of synthesis at origins, elongation on the leading strand and lagging strands and replacement of RNA primers with DNA. Current models suggest that more than one DNA polymerase is required for replication, that the proteins that start or finish chains may be different than those that perform the bulk of chain elongation, and that the proteins that replicate the leading and lagging strands may be different. To determine whether these differences result in different replication error rates, we are examining the fidelity of DNA synthesis catalyzed by eukaryotic DNA polymerases alpha, beta, delta, epsilon and gamma and the fidelity of bidirectional DNA replication by the multiprotein replication apparatus in extracts of human HeLa cells. We have found that the DNA polymerases have distinctly different error rates and specificities, which have implications for their roles in the various stages of DNA replication. Also, although the overall fidelity of replication is similar on the two strands, base substitution and frameshift error rates do differ at some sites for the leading and lagging strand replication apparatus. Finally, in order to better understand the effects of known mutagens and carcinogens on the fidelity of DNA synthesis, we are performing studies of replication fidelity with DNA molecules containing defined lesions, including psoralen monoadducts, UV photoproducts and AAF adducts. We intend to continue these studies to understand the fidelity of each of the polymerization reactions required for complete replication of the human genome.