DNA REPLICATION FIDELITY

工作总结：这个项目的长期目标是了解多蛋白复制复合体的DNA复制保真度。今年，在三个领域取得了进展。我们花费了相当大的努力来撰写和发表一篇关于DNA复制的保真度的广泛综述，从DNA聚合酶的不断演变的结构信息中进行解释。我们成功地建立了一个模型系统，可以对根部切除修复的保真度进行调查。我们确定，由四个高纯度的重组人类蛋白催化的Gou错配修复效果良好，表明修复保真度不高。根据这一新信息，我们现在正在研究可以解释基因组稳定性的假说。我们还继续研究了五种不同酵母DNA聚合酶活性部位的氨基酸替换对体内基因组稳定性的影响。丙氨酸取代了酪氨酸，假设其占据的位置在结构和功能上与DNA聚合酶I活性部位的α螺旋上的酪氨酸相同，而α螺旋对复制保真度至关重要。酵母多聚酶突变体获得了有趣的表型，包括增强或降低了基因组稳定性，以及对DNA损伤剂的敏感性改变。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，并将通过尝试纯化和表征突变聚合酶来补充。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，因为自发的和DNA损伤诱导的复制错误可能是可能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.