This research aims to understand the effects of transcription and the role of gene amplification on the gene-and transcribed-strand-specific repair of DNA damage induced by ultraviolet light (Uv) and chemical carcinogens- N-acetoxy-2-acetylaminofluorene (NAAAF) and benzo(a)pyrene diol epoxide (BPDE). It has been found that mammalian cells preferentially repair cyclobutane pyrimidine dimers (CPD) in the transcribed strand of active genes, however, we have found that the repair of BPDE-DNA adducts shows little, if any, gene-specific and strand-specific repair, and the repair of NAAAF-DNA adducts shows neither. We hypothesize that 1) UV irradiation and NAAAF or BPDE treatment have very different effects on gene activity, 2) transcription may specifically modify CPD to become better substrates for excision repair, (perhaps by introducing an intradimer phosphodiester bond break), and 3) any effects of gene amplification on preferential pair are largely a reflection of whether or not the majority of the amplified genes are transcriptionaliy active. To determine the effect of transcription on the modification of CPD we propose to examine the occurrence of intradimer phosphodiester bond breakage in the transcribed versus nontranscribed strand, and coding versus noncoding regions of the dihydrofolate reductase (DHFR) gene in normal human fibroblasts and repair deficient xeroderma pigmentosum groups A and D cells. We also propose to construct a circularized oligonucleotide containing a site-directed CPD with an intradimer phosphodiester bond break and to test its susceptibility to T4 endonuclease V and UVRABC nuclease incisions. We hypothesize that NAAAF and BPDE may hinder transcription more severely than UV irradiation does. To test this theory we will examine transcription inhibition by these agents by a nuclear runoff transcription assay and determine its relationship to the degree of preferential repair in DHFR gene. To investigate whether the preferential repair of DNA damage is temporarily coupled with transcription, we will examine repair of the c-fos and c-myc genes of Chinese hamster ovary (CHO) cells in plateau phase after induction of transcription of these genes with platelet- derived growth factors. To investigate the effects of gene amplification on the repair of carcinogen-DNA adducts, we propose to compare the repair of DNA adducts at the diploid hypoxanthine phosphoribosyl transferase gene locus with that for amplified DHFR genes in the same cells, and correlate the repair efficiency of these two genes with their transcription activities. We have found that the ERCC1CHO mutant and its parental cells repair NAAAF-DNA adducts with the same inefficient kinetics, however, the parental cells remove the adducts more efficiently in the DHFR gene. We hypothesize that CHO cells may have the capacity to repair NAAAF-DNA adducts from active genes, while ERCC1 mutant cells are deficient in this capability; other classes of ERCC mutants may deficient in different steps of repair. We will test this hypothesis by analyzing the removal of NAAAF- DNA adducts at gene level in genomic DNA as well as in ERCC mutants and wild type CHO cells.
| 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 |
