Structure-Function Studies Of DNA Replication Fidelity

今年，部分是与其他实验室合作，我们小组发表了八篇与DNA复制保真度有关的科学文章。我们发现，在真核细胞核DNA复制过程中掺入核糖核苷酸会导致发芽酵母中大量形式的染色体不稳定、杂合性丧失和非等位基因同源重组。这两种形式的不稳定性在很大程度上(如果不是完全的话)是由于DNA聚合酶epsilon结合的核糖核苷酸的拓扑异构酶1切割所致，DNA聚合酶epsilon是主要的链复制酶。我们发现DNA聚合酶ELTA在正常的DNA复制过程中主要复制滞后的DNA链，在分裂酵母中依赖同源重组的叉子重新启动后复制两条DNA链。我们表明，由核糖核苷酸还原酶突变驱动的萌芽酵母中的dNTP池失衡降低了DNA复制的保真度，这与池失衡的方式一致。我们发表了三篇文章，回顾了最近在复制保真度及其对进化和人类疾病起源的影响方面的工作。我们发表了一篇文章，重点介绍了我们监控整个萌芽酵母基因组中的核糖核苷酸掺入的方法，AWE发表了一篇文章，讨论了我们首选的真核DNA复制酶学模型与另一家实验室发表的另一种观点的关系。

This year, partly in collaboration with other laboratories, our group published eight scientific articles related to DNA replication fidelity. We showed that incorporation of ribonucleotides during eukaryotic nuclear DNA replication results in large forms of chromosomal instability in budding yeast, loss-of-heterozygosity and nonallelic homologous recombination. Both forms of instability are largely, if not exclusively, due to topoisomerase 1 incision of ribonucleotides incorporated by DNA polymerase epsilon, the primarily leading strand replicase. We showed that DNA polymerase elta, which primarily replicates the lagging strand of DNA during normal DNA replication, replicates both DNA strands after homologous recombination-dependent fork restart at a well-defined replication fork barrier in fission yeast. We showed that a dNTP pool imbalance in budding yeast that is driven by a mutation in ribonucleotide reductase reduces the fidelity of DNA replication in a manner consistent with the pool imbalance. We published three articles that reviewed recent work on replication fidelity and its implications for evolution and the origins of human diseases. We published an article highlighting our method to monitor ribonucleotide incorporation across the whole budding yeast genome, and awe published an article discussing our preferred model of eukaryotic nuclear DNA replication enzymology in relation to an alternative viewpoint published by another laboratory.