MECHANISMS OF GENOME INSTABILITY

工作概述：遗传缺陷以及潜在不稳定的高危DNA基序（ARMs）可导致基因组不稳定，两者结合可导致不稳定性和人类疾病的协同增加。酵母为人类DNA代谢基因和arm的功能分析提供了一个体内试管。我们使用ARMs来解决基因组稳定性问题，并检测DNA代谢基因中的细微缺陷，原因是具有小影响的变异可能在等位基因之间，或与ARMs或与环境因素之间表现出强烈的协同相互作用。A)基本DNA聚合酶e的改变对基因组稳定性的影响。分离出一种等位基因，通过降低DNA复制保真度，特异性地增加长同核苷酸运行中的+1移码突变。与校对缺陷相结合，双突变体是鉴定出的最强聚合酶e突变体，在错配修复背景下是致命的。B)在人类复制和修复中起重要作用的5' DNA皮瓣内切酶hFEN1可以完全补充酵母无RAD27突变体。核酸酶缺陷等位基因的几种遗传效应导致分离出新的基因毒性hFEN1突变体。缺乏与PCNA相互作用的突变体RAD27/FEN1似乎对基因组稳定性影响不大；然而，它与双链断裂（DSB）修复突变体表现出协同作用。C)我们发现（通过与Kunkel实验室的IRA合作）RAD27/FEN1的一个微妙等位基因与DNA聚合酶d3 ‘- bbb50 ’外切酶缺陷（但不包括Pol d的其他结构域）之间存在强烈的负相互作用，导致超突变和重组，dsb甚至细胞死亡。这表明除了校对外，这种外切酶还有一个新的作用。D) DNA聚合酶D的微妙突变等位基因与强隐性突变基因的许多组合可以在二倍体细胞中导致强突变表型。E)与威尔逊实验室合作，我们开发了一种酵母系统来研究人类DNA聚合酶b，它可以在DNA损伤的修复中发挥重要作用。

Summary of Work: Genetic defects as well potentially unstable at-risk DNA motifs (ARMs) can cause genome instability and the combination can lead to synergistic increases in instability and disease in humans. Yeast provides an in vivo test tube for functional analysis of human DNA metabolic genes and ARMs. We used ARMs to address genome stability and to detect subtle defects in DNA metabolic genes, reasoning that variants with a small effect might exhibit strong synergistic interactions between alleles, or with ARMS or with environmental factors. A) Alterations in the essential DNA polymerase e impact on genome stability. An allele was isolated that specifically increases +1 frameshift mutations in long homonucleotide runs by lowering DNA replication fidelity. In combination with a proofreading defect the double mutant is the strongest polymerase e mutator identified and is lethal in a mismatch repair background. B) The 5' DNA flap endonuclease hFEN1, which is important for human replication and repair, could fully complement a yeast null RAD27 mutant. The several genetic effects of a nuclease-deficient allele led to the isolation of novel genotoxic hFEN1 mutants. A mutant RAD27/FEN1 that lacks interaction with PCNA appears to have little effect on genome stability; however, it exhibited synergy with double-strand break (DSB) repair mutants. C) We discovered (through an IRA collaboration with the Kunkel lab) strong negative interactions between a subtle allele of RAD27/FEN1 and defects in the DNA polymerase d 3'->5' exonuclease (but not in other domains of Pol d) that caused hyper mutation and recombination, DSBs and even cell death. This demonstrates a novel role for this exonuclease in addition to proofreading. D) Many combinations of subtle mutator alleles of DNA polymerase d combined with strong recessive mutators can result in a strong mutator phenotype in diploid cells. E) collaborating with the Wilson lab we developed a yeast system to investigate human DNA polymerase b, which can play a major role in the repair of DNA damage.