The ability of organisms to repair altered DNA is a universal phenomenon that evolved to mitigate the mutagenic, carcinogenic, teratogenic, and toxic effects of damage in DNA. There are multiple repair processes that are lesion-specific and these appear to be as highly regulated in mammals as in bacteria. However, in contrast to the situation with bacteria, little is known about the molecular biology of repair genes and their regulatory mechanism in mammals because very few of these genes or their cDNAs have been cloned. N-alkylpurines, the most abundant adducts in DNA induced by simple alkylating carcinogens, are toxic and indirectly mutagenic and carcinogenic. A functional cDNA of human N-methylpurine- DNA glycosylase (MPG) responsible for repair of N-alkylpurines has recently been cloned by phenotypic rescue of MPG E. coli. It may be possible to similarly clone the human cDNAs of another critical repair protein, abasic endonuclease (AP-endo). The cDNAs of mouse MPG and AP endo could also be cloned using the human cDNA probes. The specific aims of this proposal are: (1) to clone full length human and mouse MPG cDNAs and their genes; (2) to construct a high-level expression vector for human MPG; (3) to purify human MPG and to produce a monoclonal and polyclonal antibodies for its quantitation; (4) to elucidate the structure and organization of human and mouse MPG genes and to identify their regulatory sequences; (5) to determine chromosomal locations of the human and mouse MPG genes; (6) to determine the effects of age, tissue type, and cell-cycle stage on MPG expression; (7) to investigate whether MPG is repressed or induced under certain conditions, and whether the MPG gene is induced and amplified in drug-resistant cells; (8) to determine whether its expression level is correlated with alkylating drug resistance in tumor xenografts and tissue sections; (9) to investigate the modulation of MPG expression during gametogenesis and embryo development in mouse; and (10) to clone human AP-endo cDNAs and characterize the human and mouse AP-endo genes. The long-range objectives of this proposal are to comprehensively characterize the genes responsible for repair of alkylation damage in humans and to elucidate the molecular basis of their regulation. Mouse provides the model system where the role of alkylation damage in mutagenesis, teratogenesis, and carcinogenesis can be studied in transgenic animals.
NIH ExPorter
NIH ExPorter