Mechanisms of Genome Instability

基因组不稳定的机制

• 批准号: 6535113
• 负责人: MICHAEL A RESNICK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6535113
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; enzyme activity; frameshift mutation; genetic recombination; genome; human genetic material tag; nucleic acid repetitive sequence; nucleic acid structure; tissue /cell culture

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.

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。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。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δ 3‘- bbb50 ’外切酶缺陷之间存在强烈的负相互作用。我们描述了Pol δ的3‘- >5’外显子作为Rad27/Fen1 5'-flap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基序的Pol δ突变结合时是致命的，这些突变使其外切酶失活，但与Pol δ其他部分的突变结合时是存活的。rad27-p等位基因本身几乎没有表型效应，但与Pol δ Exo I和Exo II基序的突变结合时也是致命的。然而，rad27-p Pol δ Exo III双突变体是存活的。它们在CAN1重复突变、染色体内和染色体间重组方面表现出强烈的协同性增加，并且需要野生型双链断裂修复基因RAD50、RAD51和RAD52来维持生存。观察到的效果与缺失后链5'瓣的rad27-null突变体相似。这些结果表明，Pol δ的3'- >5' Exo活性与Rad27/Fen1是冗余的，可以在相邻的Okazaki片段之间产生可连接的缺口，可能是通过减少滞后链中的链位移量。