Inflammation as a Mediator of Dynamic DNA Mutations

描述(由申请人提供)：这项工作的目标是确定炎症反应或其他环境损害来源造成的DNA损伤导致动态DNA突变的机制。动态突变的特征是三重重复序列的扩展，如(CAG)n/(CTG)n，已知是几种神经退行性疾病的主要致病特征。例如，在正常人的亨廷顿蛋白基因中存在5-35个重复，而在亨廷顿病(HD)患者中有40多个重复。使用HD的小鼠模型，其他实验室最近的工作表明，氧化DNA损伤产物7，8-二氢-8-氧鸟嘌呤(8-oxoG)参与了动态突变。此外，DNA修复酶OGG1也与动态突变有关，OGG1已知能从DNA双链中移除8-oxoG。我们的中心假设是基于我们的初步数据，即(CAG)n和(CTG)n序列采用非B构象，相对于双链，它们对DNA损伤高度敏感，包括8-oxoG的形成。除了含有DNA损伤的热点外，我们还发现这些非B构象不能被OGG1有效地修复。我们推测，OGG1-DNA错误修复复合体的形成稳定了非B构象，然后DNA被错误复制，聚合酶结合了多余的三联体重复。这项提议勾勒出了四个具体目标，以检验我们的假设。首先，建议进行研究以确定易于发生动态突变的重复CAG/CTG序列家族的构象。这些实验将用寡核苷酸底物和核小体核心颗粒(NCP)中的DNA进行，核小体核心颗粒(NCP)模拟染色质中DNA的包装。其次，将使用过氧亚硝酸盐作为损伤剂来建立三重重复DNA的损伤模式。过氧亚硝酸盐是炎症过程中产生的活性物种，已知能将G转化为8-oxoG。第三，OGG1介导的含有8-oxoG的DNA底物的修复，包括寡核苷酸和NCP，将被表征。这项建议的最终目的是定义复制机制对含有损伤的CAG/CTG序列的处理，并阐明OGG1在调节复制保真度中的作用。

DESCRIPTION (provided by applicant): The goal of this work is to define the mechanism by which DNA damage resulting from an inflammatory response or other environmental sources of damage contribute to dynamic DNA mutations. Dynamic mutations are characterized by the expansion of a triplet repeat sequence such as (CAG)n/(CTG)n and are known to be the primary pathogenic signature of several neurodegenerative disorders. As one example, 5-35 repeats are present in the huntingtin gene of normal individuals but there are more than 40 repeats in Huntington's disease (HD) patients. Using a mouse model of HD, recent work from other laboratories has implicated the oxidative DNA damage product 7,8-dihydro-8-oxoguanine (8-oxoG) in dynamic mutations. Furthermore, the DNA repair enzyme OGG1, which is known to remove 8-oxoG from DNA duplexes, has also been linked to dynamic mutations. Our central hypothesis is based on our preliminary data that (CAG)n and (CTG)n sequences adopt non-B conformations that are hyper-susceptible to DNA damage relative to duplex, including the formation of 8-oxoG. In addition to containing hot spots for DNA damage we have also found that these non-B conformations are not efficiently repaired by OGG1. We postulate that formation of an OGG1- DNA misrepair complex stabilizes the non-B conformation and the DNA is then misreplicated and polymerase incorporates excess triplet repeats. This proposal outlines four specific aims to test our hypothesis. First, studies are proposed to identify the conformation of a family of repetitive CAG/CTG sequences that are prone to dynamic mutations. These experiments will be performed both with oligonucleotide substrates and also DNA in nucleosome core particles (NCP) which model the packaging of DNA in chromatin. Second, the patterns of damage in the triplet repeat DNA will be established using peroxynitrite as the damaging agent. Peroxynitrite is the reactive species produced during inflammation and is known to convert G to 8-oxoG. Third, the OGG1-mediated repair of 8-oxoG-containing DNA substrates, both oligonucleotide and NCP, will be characterized. The final aim of this proposal is to define the processing of damage-containing CAG/CTG sequences by the replication machinery and elucidate the role of OGG1 in modulating replication fidelity.