DNA REPLICATION FIDELITY

工作概述：本项目的长期目标是通过多蛋白复制复合体了解DNA复制保真度。今年，在三个方面取得了进展。我们花了相当多的精力撰写并发表了一篇关于DNA复制保真度的广泛综述，该综述是通过DNA聚合酶的进化结构信息来解释的。我们成功地建立了一个模型系统，将允许调查的保真度的基础切除修复。我们确定由四种高度纯化的重组人蛋白催化的GoU错配修复效果良好，这表明修复保真度不高。我们现在正在研究可以根据这些新信息解释基因组稳定性的假设。我们还继续研究了五种不同酵母DNA聚合酶的推定活性位点的氨基酸取代对体内基因组稳定性的影响。假设丙氨酸取代了酪氨酸，在结构和功能上与DNA聚合酶I活性位点上的α螺旋上的酪氨酸相同，这对复制保真度至关重要。酵母聚合酶突变体获得了有趣的表型，包括增强或降低基因组稳定性和改变对DNA损伤剂的敏感性。这些表型取决于五种聚合酶中的哪一种被修饰。对这些表型的进一步研究正在进行中，并将通过纯化和表征突变聚合酶的尝试加以补充。这些关于基因组如何有效和正确地复制和修复的研究对人类健康很重要，因为自发和DNA损伤引起的复制错误很可能是引发人类疾病的突变的来源。

Summary of Work: The long-term goal of this project is to understand DNA replication fidelity by multiprotein replication complexes. This year, progress was in three areas. We spent considerable effort to write and publish an extensive review on the fidelity of DNA replication as interpreted from evolving structural information on DNA polymerases. We successfully established a model system that will allow investigation of the fidelity of base excision repair. We determined that repair of a GoU mismatch that is catalyzed by four highly purified recombinant human proteins works well and indicates that repair fidelity is not high. We are now investigating hypotheses that could explain genome stability in light of this new information. We also continued studies to examine the effects on genome stability in vivo of amino acid substitutions in the putative active sites of five different yeast DNA polymerases. Alanines were substituted for tryrosines hypothesized to occupy positions that are structurally and functionally equivalent to a tyrosine on an alpha helix at the active site of DNA polymerase I that is critical for replication fidelity. Interesting phenotypes have been obtained with the yeast polymerase mutants, including either enhanced or reduced genome stability and altered sensitivity to DNA damaging agents. These phenotypes depend on which of five polymerases is modified. Further investigation of these phenotypes is in progress, and will be complemented by attempts to purify and characterize the mutant polymerases. These studies of how genomes are efficiently and correctly replicated and repaired are important for human health because spontaneous and DNA damage-induced replication errors are likely sources of mutations that may initiate human diseases.