Repair of Mutagenic 8-Oxoguanine in Mammalian Genomes

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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.