Repair of Mutagenic 8-Oxoguanine in Mammalian Genomes

描述（由申请人提供）：活性氧（ROS）作为呼吸的副产物，在多种生理反应中不断产生，诱导致癌和致突变的DNA损伤，包括8-氧鸟嘌呤（8-oxoG或G*），并调节维持体内平衡的信号通路。由于其易于与A（和G）错配，G*可能是最重要的诱变损伤，通过碱基切除修复（BER）途径修复，由8-oxoG-DNA糖基化酶（OGG）切除开始。真核生物中主要的OGG （OGG1）和非同源的大肠杆菌中主要的OGG （MutM）都倾向于G*. c对作为底物。我们认为OGG I负责8-oxoG的全球基因组修复，另一个OGG OGG2优先修复G*中的G*。G或G*。A对和氧化嘧啶，负责G*的转录和复制偶联修复。这一假设得到了OGGI零细胞中转录DNA中G*的正常修复，而不是全基因组中G*的正常修复的支持。最近发现的三个人mrna序列与大肠杆菌Nei（具有ogg2型活性）和MutM具有同源性，我们将其命名为NEH 1-3。我们在NEH1中存在OGG2活性的初步证据证明了其在G*和氧化嘧啶修复中的作用的进一步表征。我们还在体内鉴定了两种共价修饰的OGGI形式，其生理功能有待研究。该项目的总体目标是全面检查哺乳动物细胞中的G*修复，其中可能涉及不同的ogg和修复复合物，用于全球基因组修复与转录活性和复制DNA的修复。目的1是表征NEH1/ OGG2，并通过x射线晶体学、生化研究、与其他BER蛋白协调修复的相互作用以及在复制和转录偶联修复中的潜在作用来阐明其结构、酶活性和体内功能。目的2是表征OGG1的修饰形式，鉴定修饰酶，并研究这种修饰的生理意义。目的3是产生NEHI零突变小鼠和细胞系，以测试NEH1在氧化碱基修复中的作用，特别是在转录活性和复制DNA中。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS), generated continuously as by-products of respiration, and during multiple physiological responses, induce carcinogenic and mutagenic DNA lesions including 8-oxoguanine (8-oxoG or G*), and regulate signaling pathways for maintaining homeostasis. Due to its propensity to mispair with A (and G), G* may be the most important mutagenic lesion which is repaired via the base excision repair (BER) pathway, initiated with its excision by 8-oxoG-DNA glycosylase (OGG). Both the major eukaryotic OGG, OGG1, and nonhomologous, major E. coli OGG, MutM, prefer the G*.C pair as substrate. We propose that OGG I is responsible for global genomic repair of 8-oxoG and a second OGG, OGG2, with preference for G* in G*.G or G*.A pairs and for oxidized pyrimidines, is responsible for transcription- and replication-coupled repair of G*. This hypothesis is supported by the normal repair of G* in transcribed DNA but not total genome in OGGI null cells. Three recently identified human mRNAs have sequence homology with E. coli Nei (with OGG2-type activity) and MutM which we named NEH 1-3. Our preliminary evidence for the presence of OGG2 activity in NEH1 warrants further characterization of its role in G* and oxidized pyrimidine repair. We have also identified two covalently modified forms of OGGI in vivo, whose physiological functions need to be examined. The overall objective of this project is a comprehensive examination of G* repair in mammalian cells which may involve distinct OGGs and repair complexes for global genome repair vs. repair in transcriptionally active and replicating DNA. Aim 1 is to characterize NEH1/ OGG2, and elucidate its structure, enzymatic activity and in vivo functions by X-ray crystallography, and biochemical studies, interaction with other BER proteins for coordination of repair, and potential role in replication and transcription-coupled repair. Aim 2 is to characterize the modified forms of OGG1, identify the modifying enzymes, and investigate the physiological significance of such modifications. Aim 3 is to generate NEHI null mutant mice and cell lines there from, to test the role of NEH1 in repair of oxidized bases specifically in transcriptionally active and replicating DNA. These comprehensive studies should provide definitive insights into various antimutagenic processes for 8-oxoG and other base lesions for prevention of radiation and ROS-induced carcinogenesis.