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期间持续产生，诱导致癌和致突变DNA损伤，包括8-氧代鸟嘌呤（8-oxoG或G*），并调节维持体内平衡的信号传导途径。由于其与A（和G）错配的倾向，G* 可能是最重要的诱变损伤，其通过碱基切除修复（BER）途径修复，由8-oxoG-DNA糖基化酶（OGG）切除启动。主要的真核细胞OGG，OGG 1和非同源的主要E. coliOGG、MutM等菌株更喜欢G*.C对作为底物。我们提出，OGG I负责8-oxoG的全局基因组修复，第二个OGG，OGG 2，优先选择G*.G或G*.A对中的G* 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缺失细胞中转录DNA中G* 的正常修复而不是总基因组中G * 的正常修复的支持。最近鉴定的三种人类mRNA与大肠杆菌具有序列同源性。coliNei（具有OGG 2型活性）和MutM，我们将其命名为NEH 1-3。我们的初步证据OGG 2活性NEH 1的存在，保证其在G* 和氧化嘧啶修复的作用进一步表征。我们还确定了两种共价修饰形式的OGGI在体内，其生理功能需要进行检查。该项目的总体目标是全面检查哺乳动物细胞中的G* 修复，其可能涉及不同的OGG和修复复合物，用于全局基因组修复与转录活性和复制DNA中的修复。目的1是表征NEH 1/OGG 2，并通过X射线晶体学和生物化学研究阐明其结构，酶活性和体内功能，与其他BER蛋白的相互作用以协调修复，以及在复制和转录偶联修复中的潜在作用。目的二是对OGG 1的修饰形式进行表征，鉴定修饰酶，并探讨这些修饰的生理意义。目的3是产生NEH 1无效突变小鼠及其细胞系，以测试NEH 1在转录活性和复制DNA中特异性地修复氧化碱基中的作用。 这些全面的研究应提供明确的见解，各种抗突变过程的8-oxoG和其他基础病变，以防止辐射和ROS诱导的致癌作用。

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.