The proposed research aims to determine the genotoxic and mutational effects of chemical carcinogen-induced DNA damage and to study the chemistry of their repair. Using a viral-DNA transfection system and an in vitro incision assay we have found that the E. coli, uvrA, B and C gene products initiate the repair of a wide variety of lesions induced by ultraviolet light (UV), chemical carcinogens, such as N-acetoxy-2-acetylaminofluorene (NAAAF), N-hyroxy-2-acetylaminofluorene (N-OH-AF), benzo(a)pyrene diol-epoxide (BPDE), and DNA minor groove binding agents sucha as anthramycin and cc-1065. These lesions have different effects on DNA structure; the purified UvrA, B, and C proteins repair them with varying degrees of efficiency. Furthermore, helicase II has different effects on UvrABC activity in repairing these damages. We will determine the biochemcial basis of these effects. Using the M13mp10 Lac+ yields LAC - forward mutation system we have detected a broad spectrum of mutations and recombinations induced by AAF- and AF-DNA aducts. DNA sequence changes in these mutants show that 1) there are sequences in which both adducts induce identical base changes, and 2) SOS repair may greatly alter the mutation spectrum induced by DNA adducts. We shall determine the effects that adduct formation and DNA sequence have on DNA replication, repair susceptibility and mutagenesis and apply our findings to studying the molecular details of mutations induced by three BPDE isomers which we have found to have different genotoxicities and mutagenicities. Using purified UvrA, B and C proteins and helicase II, in combination with gene probes and Southern blot techniques, we have developed a technique to detect chemically induced DNA damage in the aprt and dhfr genes in Chinese hamster ovary (CHO) cells and found that in the aprt gene AF adducts occur preferentially in noncoding sequences. We propose to use this method to measure differential induction and/or repair in carcinogen treated mammalian cells (both wild type and hypersensitive mutants), to study the role of gene activity (transcription, amplification and methylation) in DNA damage and repair, and to assess the role of differential repair in cytotoxicity and mutation.
| Denissenko, M F; Chen, J X; Tang, M S et al. (1997) Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc Natl Acad Sci U S A 94:3893-8 |
