Ubiquitous repair mechanisms have evolved to remove DNA lesions, (both endogenous and those induced by genotoxic agents), that have been implicated in mutagenesis, cancer, and aging. Small mutagenic alkyl and cyclic ethenobase adducts induced by simple carcinogens, such as vinyl chloride and antitumor drugs, e.g., cyclophosphamide, are repaired via the base excision repair (BER) pathway; its first step is removal of the adduct by N-methylpurine-DNA glycosylase (MPG), generating abasic (AP) sites. In mammalian cells, only one MPG with a wide substrate range has so far been characterized. Furthermore, AP sites, also generated spontaneously and by other DNA glycosylases, are themselves cytotoxic and mutagenic; the first step tin their repair is DNA strand cleavage by AP-endonucleases (APE). The major mammalian APE (APE-1) also activates transcription factor AP-1 and is itself a repressor in Ca2+-dependent gene regulation. The molecular mechanisms of mammalian MPGs and APEs are not known. In particular, the mechanism of MPG, with a broad substrate range, should be significantly different from that of the well-characterized uracil-DNAglycosylase. The availability of molecular probes for human and mouse MPGs and APEs provides a unique opportunity for a comprehensive investigation of their reaction mechanisms and in vivo functions.
The specific aims of this continuing project are: (1) to characterize the mechanisms of lesion recognition and catalysis by mammalian MPG's; (2) to elucidate the mechanisms of multiple functions of human APE-1; (3) to test the hypothesis that BER proteins form a repairosome complex; (4) to isolate MPG-negative mice and cell lines in order to evaluate the etiologic role of specific lesions in tumorigenesis, mutagenesis and toxicity; and (5) to isolate APE-1 negative human cells for elucidating the role of AP sites in spontaneous and induced mutations. The long range objectives of this project are to characterize the repair proteins responsible for repairing alkylation and oxidative damage in mammalian genomes, and their regulation, and eventually to develop approaches for deliberately modulating repair in normal and tumor cells.
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