Mechanisms Of Genome Instability

基因组不稳定的机制

• 批准号: 7007437
• 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/7007437
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; biomarker; cadmium; enzyme activity; frameshift mutation; gene environment interaction; genetic recombination; genome; human genetic material tag; ionizing radiation; neoplasm /cancer genetics; nucleic acid repetitive sequence; nucleic acid structure; tissue /cell culture

Sensitized genetic systems and DNA at-risk motif reporters have been developed through several years of investigating genetic risks and synergistic interactions with DNA metabolic defects. These two methodologies allow us to identify the strongest cases of genome instability and to understand molecular mechanisms underlying the instability. Repeat motifs have been identified in many organisms that are at high risk for genetic change in wild-type or mutation-prone cells. They can be a source genomic instability, cancer and in some cases provide changes that extend the host range of infectious microbes. We surmised from our various experimental approaches that many of these At Risk Motifs (ARMs) can form non-canonical DNA structures that are poor substrates for replication or post-replication repair. This led us to propose that ARMs can be a major source of genome instability. Once the underlying mechanisms are unraveled, ARMs also provide a powerful tool of discovery in mechanistic studies of DNA metabolism. Using a variety of genetic and molecular approaches with yeast, we have characterized several types of ARMs and employed them to address mechanisms of genome instability. These include small direct repeats separated by up to 100 bases, a motif common in all organisms, long inverted repeats in configurations similar to many arrangements of the frequently occuringAlu and LINE sequences in the human genome, homonucleotide runs as short as 8 to 10 bases that can lead to extremely high levels of frameshift mutation and other types of unstable mini- and microsatellites. We have established that defects in several aspects of DNA metabolic processes greatly exacerbate the instability associated with ARMs, in essence dramatically increasing the risk associated with the at-risk motifs. DNA repair, replication and processing of DNA intermediates require coordinated interactions between many proteins. The combination of subtle changes in one or more DNA metabolic acting at ARMs can lead to synergistic increases in genome instability. We employ a variety of sophisticated genetic and biochemical approaches to identify combinations of genes that are important for maintaining genome stability. Our studies over the last several years have concentrated on the interplay between genes involved in DNA replication, double-strand break repair, mismatch repair and base excision repair in maintaining genome stability, particularly at ARM sites. We concentrated our recent research on the multiple biological roles of the Pol delta -exonuclease. For over thirty years, proofreading of errors that arise during replication was considered the only biological function of this enzymatic activity. A defect in proofreading can increase the mutation rate and lead to synthetic lethality or extreme hypermutability when combined with a defect in postreplication mismatch repair (MMR). Based on specific genetic interactions or proofreading mutants with defects in Exo1 and Rad27/Fen. along with biochemical analysis of yeast Pol ? we have discerned additional biological roles for the intrinsic exonuclease of Pol ?, specifically in the maturation of Okazaki fragments and in MMR. We have questioned whether the Pol ?-Exo performs all these biological functions?proofreading, Okazaki maturation, and MMR-- in association with the replicative complex or as an exonuclease separate from DNA replication. We identified a novel category of yeast Pol ? mutants at amino acid 523, Pol3-L523X, that are defective in processive DNA synthesis in the presence of PCNA, but only when the error rate of incorporation is high because of a dNTP imbalance. Surprisingly, the mutant enzymes retained robust 3'?5'-exonuclease activity. Based on their biochemical properties, the mutant holoenzymes appear to be impaired in switching of the nascent 3'-end of the newly synthesized DNA strand between the polymerase and the exonuclease active sites. Based on our previous studies we identified a set of characteristic in vivo genetic features that indicate strongly implicate the Pol delta in mismatch repair and in Okazaki maturation in lagging strand DNA replication. These features included synergistic interaction with defects in 5'-exonuclease EXO1 and in 5'-flap endonuclease RAD27/FEN1, as well as highly increased mutation rates of duplications flanked by short dispersed repeat ARMs. All the features of the pol3-L523X mutants, including mutation rates and spectra as well as negative synergy with defects in msh2, exo1 and rad27/fen1,were indistinguishable from previously studied Exo-defective mutants. Thus, all three biological functions of Pol ?-Exo appear to be severely impaired by the switching defect in Pol3-L523X mutations. We have concluded that all three biological functions are performed by Pol?-Exo within the replication complex during DNA replication.

敏化遗传系统和DNA危险基序报告是通过几年研究遗传风险和与DNA代谢缺陷的协同作用而发展起来的。这两种方法使我们能够识别基因组不稳定性的最强案例，并了解这种不稳定性背后的分子机制。 重复基序已经在许多生物中被发现，这些生物具有很高的野生型或易突变细胞的遗传变化风险。它们可能是基因组不稳定、癌症的根源，在某些情况下，它们提供的变化扩大了感染微生物的宿主范围。我们从各种实验方法中推测，许多处于风险中的基序(ARM)可以形成非规范的DNA结构，这些结构不利于复制或复制后修复。这导致我们提出，手臂可能是基因组不稳定的主要来源。一旦潜在的机制被解开，ARM也为DNA新陈代谢的机制研究提供了一个强大的发现工具。使用各种遗传和分子方法处理酵母，我们已经确定了几种类型的臂的特征，并利用它们来解决基因组不稳定的机制。这些重复序列包括长达100个碱基的小的直接重复序列、在所有生物体中普遍存在的基序、与人类基因组中频繁出现的Alu和Line序列的许多排列相似的长反向重复序列、短至8至10个碱基的同源核苷酸序列，这可能导致极高水平的移码突变以及其他类型的不稳定的小卫星和微卫星。我们已经确定，DNA代谢过程的几个方面的缺陷极大地加剧了与武器相关的不稳定性，本质上极大地增加了与处于危险中的基序相关的风险。 DNA中间体的DNA修复、复制和加工需要多种蛋白质之间的协调作用。一个或多个DNA代谢的微妙变化结合在一起，可能会导致基因组不稳定性的协同增加。我们采用了各种复杂的遗传和生化方法来识别对维持基因组稳定很重要的基因组合。在过去的几年里，我们的研究集中在参与DNA复制、双链断裂修复、错配修复和碱基切除修复的基因之间在维持基因组稳定方面的相互作用，特别是在ARM位点。 我们最近的研究集中在POL增量核酸外切酶的多种生物学作用上。三十多年来，对复制过程中出现的错误进行校对被认为是这种酶活性的唯一生物学功能。校对的缺陷会增加突变率，当与复制后错配修复(MMR)的缺陷结合在一起时，会导致合成致死性或极端的高度变异性。根据特定的遗传交互作用或校对带有Exo1和Rad27/Fen缺陷的突变体。结合酵母菌Pol？我们已经发现了POL？内在核酸外切酶的其他生物学作用，特别是在冈崎片段的成熟和MMR中。 我们质疑Pol？-Exo是与复制复合体结合还是作为一种独立于DNA复制的核酸外切酶来执行所有这些生物学功能？校对、Okazaki成熟和MMR。我们鉴定了一种新的酵母菌Pol？位于氨基酸523，Pol3-L523X的突变体，在增殖细胞核抗原存在的情况下，在进行性DNA合成中存在缺陷，但只有在由于dNTP失衡而导致掺入错误率较高的情况下才有缺陷。令人惊讶的是，突变的酶保持了很强的3‘？5’-核酸外切酶活性。根据它们的生化特性，突变的全酶似乎在新合成的DNA链的新的3‘端在聚合酶和核酸外切酶活性位点之间的切换中受到损害。基于我们以前的研究，我们在体内发现了一组特征的遗传特征，这些特征表明Pol Delta与错配修复和Okazaki成熟与滞后的链DNA复制密切相关。这些特征包括与5‘-核酸内切酶EXO1和5’-翻盖内切酶RAD27/FEN1缺陷的协同作用，以及短分散重复臂两侧重复序列突变率的显著增加。Pol3-L523X突变体的所有特征，包括突变率和突变谱，以及与MSH2、Exo1和rad27/fen1缺陷的负协同作用，都与以前研究的外源缺陷突变体没有区别。因此，Pol3-L523X突变的开关缺陷似乎严重损害了Pol？-Exo的所有三种生物学功能。我们的结论是，在DNA复制过程中，所有这三种生物学功能都是由复制复合体中的POL？-Exo执行的。