Repair of Mutagenic 8-Oxoguanine in Mammalian Genomes

描述(申请人提供)：呼吸副产物、在多种生理反应过程中持续产生的活性氧物种(ROS)可诱发致癌和致突变的DNA损伤，包括8-oxoguanine(8-oxoG或G*)，并调节维持内环境稳定的信号通路。由于G*容易与A(和G)错配，G*可能是最重要的突变损伤，它通过碱基切除修复(BER)途径修复，首先被8-oxoG-DNA糖基酶(OGG)切除。主要的真核生物Ogg OGG1和非同源的主要E.ColiOgg MutM都喜欢G*.C对作为底物。我们认为OGG I负责8-oxoG的全局基因组修复，第二个OGG OGG2优先于G*.G或G*中的G*，A负责G*的转录和复制偶联修复。这一假说得到了转录DNA中G*的正常修复而不是OGGI缺失细胞中全基因组的支持。最近发现的三个人mRNAs与E.ColiNei(具有OGG2型活性)和MutM具有序列同源性，我们将其命名为NEH1-3。我们的初步证据表明，在NEH1中存在OGG2活性，这为进一步确定其在G*和氧化嘧啶修复中的作用提供了依据。我们还在体内鉴定了两种共价修饰的OGGI形式，其生理功能需要检测。这个项目的总体目标是全面检查哺乳动物细胞中的G*修复，这可能涉及不同的OGG和修复复合体，用于全球基因组修复与转录活性和复制DNA的修复。目的1鉴定NEH1/OGG2，并通过X射线结晶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2是表征OGG1的修饰形式，鉴定修饰酶，并研究这些修饰的生理意义。目的3是建立NEHI缺失突变小鼠和细胞系，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。 这些全面的研究应该为8-oxoG和其他碱基损伤的各种抗突变过程提供明确的见解，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.