Oxidatively induced DNA lesions have been implicated in the etiology of many diseases (including cancer), and in aging. Among the many base lesions induced by reactive oxygen species (ROS), 5-hydroxyuracil (5- OHU) and 8-oxoguanine (8-oxoG) are thought to be responsible for the majority of ROS-induced GC to AT and GC to TA mutations, respectively. Repair of oxidatively damaged bases in all organisms occurs primarily via the DNA base excision repair (BER) pathway, initiated with excision by DNA glycosylases. Only two previously characterized mammalian DNA glycosylases, OGG1 and NTH1, are thought to excise most of the oxidative damage. However, the lack of phenotype and continued repair of 8-oxoG and thymine glycol from the transcribed DNA sequences of OGG1- and NTH1-null mouse cells suggested the presence of additional glycosylases. We have recently discovered and partially characterized two human orthologs of E.coli Nei, and named them NElL (Nei-like)-1 and 2. They are unique in that, unlike most DNA glycosylases which are active only with duplex DNA, NEILs excise lesions from bubble DNA. NEIL2 has a 4- to 5-fold higher affinity and activity for 5-OHU in bubble vs duplex DNA, and it excises 8-oxoG from bubble but not duplex DNA. These observations strongly suggest that NEIL2 plays a major role in repairing lesions in transcription bubbles, and so contributes to the transcription-coupled base excision repair (TC-BER) observed in OGG1-null cells. The stable interaction of NEIL2 with RNA Polymerase II (Pol II) and heterogeneous nuclear ribonucleo protein (hnRNP-U), and the significant increase in endogenous mutations in NEIL2-deficient cells, are consistent with this hypothesis, hnRNP-U, an abundant multifunctional nuclear matrix protein, has also been shown to regulate transcription. NEIL2 carries out a beta-delta-reaction like E. coli MutM/Nei, generating a 3'-P which is removed by polynucleotide kinase (PNK), but not by AP-endonuclease. The discovery of NEIL2's strong preference for bubble DNA, and its association with Pol II and hnRNP-U and a novel repair pathway that is APE1-independent but PNK-dependent, have set the stage for developing a comprehensive picture of how 5- OHU, 8-oxoG and other mutagenic lesions, are repaired in transcribed vs. nontranscribed regions of the genome, and how NEIL2-mediated repair involves specific interactions with hnRNP-U and Pol II. A variety of techniques, including recombinant DNA technology, enzyme kinetics, coimmunoprecipitation experiments to examine protein-protein irteractions, and finally siRNA technology, will be used to pursue the following aims, to: (1) elucidate the mechanistic basis by which NEIL2's activity is stimulated by hnRNP-U in bubble DNA; (2) test the hypothesis that NEIL2-mediated strand incision leads to transcriptional arrest; and (3) test the hypothesis that NEIL2 is involved in PNK-dependent TC-BER. The long-term goal of our research is to elucidate the mechanistic basis of TC-BER of oxidized bases, which should provide definitive insights into cellular responses to the genotoxic effects of the oxidized bases and help develop strategies for better prevention or treatment of radiation- and ROS-induced carcinogenesis.
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