Reactive oxygen species (ROS), endogenously generated as respiration by-products and exogenously by radiation and other genotoxins, induce a variety of base damage and DNA strand breaks in cell genomes, which are mostly repaired via the base excision repair (BER) pathway, initiated with excision of base lesions by DNA glycosylases. Accumulation of oxidized base damage without a strong phenotype in mice lacking OGG1 and NTH1, the major mammalian glycosylases, suggested that additional glycosylases repair active genomic sequences. In our previous project period we characterized human glycosylases (NEIL1 and NEIL2) with overlapping substrate preferences as OGG1 and NTH1. However, only the NEILs excise lesions from single-stranded or bubble DNAs. Stable interaction of NEIL2 with RNA polymerase II, and of NEIL1 with the sliding clamp PCNA, and other DNA replication-associated proteins led us to hypothesize a preferential role for NEIL1 and -2 in replication (RAR)- or transcription-associated repair, respectively. We further postulate that NEIL1 preferentially repairs base damage in leading or lagging template strand (preplicative repair), and also removes incorporated base lesion in the nascent DNA strand (post-replicative repair). Formation of multiprotein complexes in response to radiation and oxidative stress, which may also be affected by covalent modification of component proteins, led us to hypothesize that distinct repair complexes are assembled for repairing endogenous vs. radiation/ROS-induced damage, and in G1 vs. S-phase cells, by utilizing distinct DNA polymerases and other proteins including RPA, FEN1 (activated by PCNA) and Werner or Bloom protein with DNA helicase activity. In this competing renewal project we will examine various facets of this hypothesis by testing whether (1) NEIL1 forms distinct repair complexes in G1 vs. S-phase cells, and in response to oxidative stress;(2) NEIL1 preferentially repairs base damage in both template and nascent DNA during replication by interacting with the replicating complex;(3) damage in replicating plasmid is preferentially repaired in vivo relative to nonreplicating plasmid;and (4) NEIL1-induced cleavage at the damage site of the replicating DNA strand leads to double-strand breaks. Health Relevance: Successful completion of these studies will not only establish the novel concept of RAR, but will also illuminate various interactions among repair proteins distinct complexes. Such basic understanding of interaction interfaces in repair complexes could be exploited in developing therapeutic intervention strategies for sensitizing tumor cells or protecting healthy cells during chemo/radiation therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Radiation Therapeutics and Biology Study Section (RTB)
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Okano, Paul
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University of Texas Medical Br Galveston
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Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M et al. (2013) The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions. J Mol Biol 425:2359-71
Hegde, Muralidhar L; Hegde, Pavana M; Bellot, Larry J et al. (2013) Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins. Proc Natl Acad Sci U S A 110:E3090-9
Della-Maria, Julie; Hegde, Muralidhar L; McNeill, Daniel R et al. (2012) The interaction between polynucleotide kinase phosphatase and the DNA repair protein XRCC1 is critical for repair of DNA alkylation damage and stable association at DNA damage sites. J Biol Chem 287:39233-44
Hegde, Muralidhar L; Izumi, Tadahide; Mitra, Sankar (2012) Oxidized base damage and single-strand break repair in mammalian genomes: role of disordered regions and posttranslational modifications in early enzymes. Prog Mol Biol Transl Sci 110:123-53
Boldogh, Istvan; Hajas, Gyorgy; Aguilera-Aguirre, Leopoldo et al. (2012) Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine. J Biol Chem 287:20769-73
Dey, Sanjib; Maiti, Amit K; Hegde, Muralidhar L et al. (2012) Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2. DNA Repair (Amst) 11:570-8
Hegde, Muralidhar L; Mantha, Anil K; Hazra, Tapas K et al. (2012) Oxidative genome damage and its repair: implications in aging and neurodegenerative diseases. Mech Ageing Dev 133:157-68
Mandal, Santi M; Hegde, Muralidhar L; Chatterjee, Arpita et al. (2012) Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3'-phosphatase in maintenance of mitochondrial genome. J Biol Chem 287:2819-29
Hegde, Muralidhar L; Hegde, Pavana M; Arijit, Dutta et al. (2012) Human DNA Glycosylase NEIL1's Interactions with Downstream Repair Proteins Is Critical for Efficient Repair of Oxidized DNA Base Damage and Enhanced Cell Survival. Biomolecules 2:564-78
Hegde, Muralidhar L; Banerjee, Srijita; Hegde, Pavana M et al. (2012) Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction. J Biol Chem 287:34202-11

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