Mitochondria and Mutagenesis

描述（由申请人提供）：线粒体功能障碍是癌症的标志之一。然而，关于线粒体功能障碍是如何导致致癌的，我们知之甚少。我们在这项提议中描述的研究表明，线粒体在保护细胞免受核基因组突变（人类癌变的一个重要事件）中的新作用。我们以酿酒酵母为模型生物，分析了线粒体功能破坏对核基因组突变的影响。我们测量了油菜耐药菌落的频率，作为核基因组突变的一个指标。我们的数据表明，线粒体功能障碍导致核基因组突变(i)在缺乏整个线粒体基因组（rho¿或p¿）或线粒体DNA缺失（rho-或p-）（线粒体遗传功能障碍，MGD）的突变菌株中（ii）当抗霉素A阻断野生型酵母的氧化磷酸化（线粒体代谢功能障碍，MMD）时。MMD和MGD细胞的核突变频率分别高于未处理的对照和野生型细胞。MGD导致细胞内ROS水平降低。相反，烟雾导致细胞内活性氧（ROS）水平升高。我们证明了由MGD引起的核基因组突变依赖于REV1、REVS或REV7基因产物，这些基因产物都与复制后修复（PRR）有关。然而，由烟雾病引起的核基因组突变不涉及REV1、REVS或REV7基因。此外，我们提供的证据表明，Rtg2蛋白（逆行2）参与线粒体到细胞核的逆行反应途径保护细胞免受核基因组突变。基于这些观察，我们假设线粒体保护并使用多种途径来保护核基因组免受突变。我们提出了4个具体目标来检验所提出的假设。在所有情况下，我们都有初步数据，为拟议的具体目标提供基础。这些具体目的是：1)建立线粒体到细胞核逆行反应途径在线粒体功能障碍引起的核基因组突变中的作用2)确定氧化应激反应受损是否导致线粒体功能障碍引起的核基因组突变3)确定复制后修复途径在线粒体功能障碍引起的核基因组突变中的作用4)确定线粒体功能障碍引起的核基因组突变的性质我们的研究将有助于深入了解线粒体功能障碍导致核基因组突变的分子遗传机制，这对人类癌症和其他疾病具有重要意义。

DESCRIPTION (provided by applicant): Mitochondrial dysfunction is one of the hallmarks of cancer. However, very little is known about how mitochondrial dysfunction leads to carcinogenesis. Our studies described in this proposal suggest a novel role for mitochondria in protecting cells from nuclear genome mutagenesis, an important event in human carcinogenesis. Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine resistant colonies as an indicator of nuclear genome mutagenesis. Our data demonstrate that mitochondrial dysfunction leads to mutation in the nuclear genome (i) in mutant strains lacking the entire mitochondrial genome (rho¿ or p¿) or those with deleted mitochondrial DNA (rho- or p-) (mitochondrial genetic dysfunction, MGD) and (ii) when oxidative phosphorylation is blocked in wild type yeast by antimycin A (mitochondrial metabolic dysfunction, MMD). The nuclear mutation frequencies in both MMD and MGD cells were higher compared to untreated control and wild type cells respectively. MGD led to decreased intracellular levels of ROS. In contrast MMD led to increased intracellular levels of reactive oxygen species (ROS). We demonstrate that nuclear genome mutagenesis due to MGD is dependent on REV1, REVS or REV7 gene products, all implicated in post-replication repair (PRR). However, nuclear genome mutagenesis due to MMD does not involve REV1, REVS or REV7 genes. Furthermore, we provide evidence that Rtg2 protein (retrograde 2) involved in mitochondria-to-nucleus retrograde response pathway protect cells from nuclear genome mutagenesis. Based on these observations we hypothesize that mitochondria protect and employ multiple pathways to guard the nuclear genome against mutagenesis. We propose 4 specific aims to test the proposed hypothesis. In all cases we have preliminary data that provide the basis for the proposed specific aims. These specific aims are:1) Establish the role of mitochondria-to-nucleus retrograde response pathway in nuclear genome mutagenesis due to mitochondrial dysfunction 2) Determine whether compromised oxidative stress response leads to nuclear genome mutagenesis due to mitochondrial dysfunction 3) Determine the role of post-replication repair pathway in nuclear genome mutagenesis due to mitochondrial dysfunction 4) Determine the nature of nuclear genome mutagenesis due to mitochondrial dysfunction Our proposed study should provide insight into the molecular genetic mechanisms of nuclear genome mutagenesis due to mitochondrial dysfunction that is of fundamental significance to human cancer and other diseases.