MECHANISMS OF GENOME INSTABILITY

工作总结：遗传缺陷以及潜在的不稳定高危DNA基序(ARM)可导致基因组不稳定，两者结合可导致人类不稳定和疾病的协同增加。酵母为人体DNA代谢基因和手臂的功能分析提供了体内试管。我们使用ARM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，理由是影响较小的变异可能会在等位基因之间、与ARM或与环境因素之间显示出强烈的协同作用。A)基本DNA聚合酶e的改变对基因组稳定性的影响。分离出了一种等位基因，它通过降低DNA复制保真度而在长时间的同源核苷酸序列中特异性地增加+1移码突变。结合校对缺陷，双重突变体是已发现的最强的聚合酶e突变体，在错配修复背景下是致命的。B)对人类复制和修复起重要作用的5‘DNA翻盖内切酶hFEN1可以完全补充酵母缺失的RAD27突变体。核酸酶缺失等位基因的几种遗传效应导致了新的遗传毒性hFEN1突变体的分离。缺乏与增殖细胞核抗原相互作用的突变体RAD27/FEN1似乎对基因组稳定性几乎没有影响；然而，它与双链断裂(DSB)修复突变体显示出协同作用。C)我们发现(通过IRA与昆克尔实验室的合作)RAD27/FEN1的一个微小等位基因与DNA聚合酶d3‘-&gt；5’外切酶缺陷(但不在Pold的其他区域)之间存在强烈的负相互作用，从而导致超突变和重组、DSB，甚至细胞死亡。这证明了这种核酸外切酶除了校对之外还有一个新的作用。D)DNA聚合酶d的微妙突变等位基因与强隐性突变相结合的许多组合可以在二倍体细胞中产生强突变表型。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损伤的修复中发挥主要作用。

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.