DNA REPLICATION FIDELITY

工作总结：本项目的长期目标是通过多蛋白复制复合物了解DNA复制保真度。今年，在三个方面取得了进展。我们通过检测hMLH 1、hMSH 3和hMSH 6缺陷与铂-DNA加合物复制旁路之间的相关性，研究了错配修复蛋白在人肿瘤细胞系顺铂和奥沙利铂耐药性中的作用。结果支持MutS α功能缺陷而非MutS β功能缺陷导致顺铂加合物的复制旁路增加并因此导致耐药性的假设。我们开始寻找协同生存和增变因子的影响所产生的基因编码复制DNA聚合酶δ和皮瓣核酸内切酶负责处理冈崎片段的滞后链突变。我们还开始研究，以检查在酵母复制型DNA聚合酶中的特定氨基酸取代的体内效应。我们取代了酪氨酸假设占据一个结构和功能等效的位置上的α螺旋在活性位点的DNA聚合酶的pol I和Pol α家族酶与丙氨酸。我们早期的研究表明，在Klenow片段Pol I中Tyr到Ala的取代导致聚合酶保真度降低。我们现在发现，具有假定的同源多聚突变的单倍体酵母菌株是可行的。它改变了对羟基脲（复制抑制剂）和甲磺酸甲酯（DNA损伤剂）的敏感性，并具有升高的自发突变率以及增加的由羟氨基嘌呤（一种具有错误编码潜力的碱基类似物）诱导的诱变。这些表型正在进一步研究中，并将通过尝试纯化和表征突变体聚合酶来补充。这些关于基因组如何有效和正确复制的研究对人类健康非常重要，因为自发的和DNA损伤诱导的复制错误可能是引发人类疾病的突变的来源。- 辅助蛋白，DNA复制保真度，缺失，突变体，T4 DNA聚合酶

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 investigated the roles of mismatch repair proteins in the cisplatin and oxaliplatin resistance of human tumor cell lines by examining the correlation between defects in hMLH1, hMSH3, and hMSH6 and replicative bypass of platinum-DNA adducts. The results support the hypothesis that defects in MutS alpha function, but not MutS beta function, lead to increased replicative bypass of cisplatin adducts and therefore drug resistance. We began a search for synergistic survival and mutator effects resulting from mutations in the genes encoding replicative DNA polymerase delta and the flap endonuclease responsible for processing Okazaki fragments on the lagging strand. We also initiated studies to examine the in vivo effects of a specific amino acid substitution in yeast replicative DNA polymerases. We replaced a tryrosine hypothesized to occupy a structurally and functionally equivalent position on an alpha helix at the active site of DNA polymerases in the pol I and Pol alpha family enzymes with an alanine. Our earlier studies showed that a Tyr to Ala substitution in Klenow fragment Pol I resulted in a polymerase with reduced fidelity. We now find that a haploid yeast strain with the putative homologous pol epsilon mutation is viable. It has altered sensitivity to hydroxyurea (replication inhibitor) and methylmethane sulfonate (a DNA damaging agent), and has an elevated spontaneous mutation rate as well as increased mutagenesis induced by hydroxylaminopurine, a base analog with miscoding potential. These phenotypes are under further investigation and will be complemented by attempts to purify and characterize the mutant pol epsilon. These studies of how genomes are efficiently and correctly replicated are important for human health because spontaneous and DNA damage-induced replication errors are likely sources of mutations that may initiate human diseases. - Accessory Proteins, DNA Replication Fidelity, Deletions, Mutator, T4 DNA Polymerase