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。5‘DNA翻盖内切酶hFEN1对人类的复制和修复很重要，它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。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Delta3‘-&gt；5’外切酶缺陷之间存在强烈的负相互作用。我们描述了Pol Delta的3‘--&gt；5’exo作为Rad27/Fen15‘-翻盖核酸内切酶的补充或备份的新作用。酵母rad27零等位基因与Exo I、Exo II和Exo III基序中的Pol Delta突变结合在一起是致命的，这些突变使其外切酶失活，但它在Pol Delta其他部分的突变中是可行的。Rad27-p等位基因本身几乎没有表型影响，与Pol Delta Exo I和Exo II基序的突变结合在一起也是致命的。然而，rad27-p Pol Delta Exo III双突变体是可行的。它们在CAN1复制突变、染色体内和染色体间重组方面表现出强烈的协同增加，并需要野生型双链断裂修复基因Rad50、RAD51和RAD52才能生存。观察到的效果类似于在移除滞后链上的5‘瓣方面缺失的rad27缺失突变体的效果。这些结果表明，Pol Delta的3‘-&gt；5’Exo活性与Rad27/Fen1一起是多余的，因为它可能通过减少滞后链中的链位移量而在相邻的Okazaki片段之间产生可连接的缺口。

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.