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。我们通过检测hMLH1、hMSH3和hMSH6缺陷与铂- dna加合物复制旁路的相关性，研究错配修复蛋白在人肿瘤细胞系顺铂和奥沙利铂耐药中的作用。结果支持MutS α功能缺陷导致顺铂加合物复制旁路增加的假设，而不是MutS β功能缺陷，从而导致耐药。我们开始寻找由编码复制DNA聚合酶delta和负责处理滞后链上冈崎片段的皮瓣内切酶的基因突变引起的协同生存和突变效应。我们还开始研究酵母复制DNA聚合酶中特定氨基酸替代的体内效应。我们用丙氨酸取代了假设在pol I和pol α家族酶的DNA聚合酶的α螺旋上占据结构和功能等效位置的tryrosine。我们早期的研究表明，在Klenow片段Pol I中Tyr到Ala的替换导致聚合酶的保真度降低。我们现在发现一个单倍体酵母菌株假定同源的pol ε突变是可行的。它改变了对羟基脲（复制抑制剂）和甲基甲烷磺酸（一种DNA损伤剂）的敏感性，并且具有较高的自发突变率以及羟胺嘌呤（一种具有错误编码潜力的碱基类似物）诱导的诱变增加。这些表型正在进一步研究中，并将通过纯化和表征突变pol epsilon的尝试加以补充。这些关于基因组如何有效和正确地复制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。-辅助蛋白，DNA复制保真度，缺失，突变，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