Mechanisms of Genome Instability

工作总结：遗传缺陷以及潜在的不稳定风险DNA基序（ARM）可导致基因组不稳定性，并且组合可导致人类不稳定性和疾病的协同增加。酵母为人类DNA代谢基因和ARM的功能分析提供了体内试管。5'端DNA侧翼核酸内切酶hFEN 1对人类复制和修复非常重要，可以完全补充酵母缺失的RAD 27突变体。核酸酶缺陷等位基因的几个遗传效应导致分离新的遗传毒性hFEN 1突变体。缺乏与PCNA相互作用的突变体RAD 27/FEN 1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。我们发现（通过伊拉与Kunkel实验室的合作）RAD 27/FEN 1的一个微妙等位基因与DNA聚合酶delta 3 '->5'外切核酸酶的缺陷之间存在强烈的负相互作用。我们描述了Pol delta的3 '->5' Exo作为Rad 27/Fen 1 5 '-flap内切核酸酶的补充或备份的新作用。酵母rad 27无效等位基因与使其核酸外切酶失活的Exo I、Exo II和Exo III基序中的Pol δ突变组合是致命的，但与Pol δ的其他部分中的突变组合是可行的。rad 27-p等位基因本身几乎没有表型效应，与Pol delta Exo I和Exo II基序中的突变组合也是致命的。然而，rad 27-p Pol delta Exo III双突变体是可行的。它们在CAN 1重复突变、染色体内和染色体间重组中表现出强烈的协同增加，并且需要野生型双链断裂修复基因RAD 50、RAD 51和RAD 52来维持生存力。观察到的效果类似于在落后链中去除5'瓣中缺陷的rad 27无效突变体的效果。这些结果表明，Pol δ的3 '->5' Exo活性对于Rad 27/Fen 1来说是多余的，用于在相邻的Okazaki片段之间产生可连接的切口，这可能是通过减少落后链中的链置换量来实现的。

Summary of Work: Genetic defects as well as 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. 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. 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 delta 3'->5' exonuclease. We described a novel role of the 3'-->5' Exo of Pol delta as a supplement or backup for the Rad27/Fen1 5'-flap endonuclease. A yeast rad27 null allele was lethal in combination with Pol delta mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol delta. The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol delta Exo I and Exo II motifs. However, rad27-p Pol delta Exo III double mutants were viable. They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability. Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5' flaps in the lagging strand. These results suggest that the 3'-->5' Exo activity of Pol delta is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments,possibly by reducing the amount of strand-displacement in the lagging strand.