ASSESSMENT OF DNA-DAMAGE AND REPAIR IN SPECIFIC GENOMIC REGIONS BY QUANTITATIVE IMMUNE-COUPLED PCR

Fine analysis of DNA damage and repair at the subgenomic level has ind icated a microheterogeneity of DNA repair in mammalian cells, includin g human. In addition to the well established Southern hybridization-ba sed approach to investigate gene-specific DNA damage and repair, alter native methods utilizing the sensitivity of PCR have been evaluated. T he latter technique has relied on decreased PCR amplification clue to damage in template DNA. We have developed a novel quantitative assay c ombining the selective recovery of DNA damage containing genomic fragm ents with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti- 9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated huma n skin fibroblasts was immunoprecipitated with polyclonal antibody BP- 1. Recovered target sequences were amplified by PCR using primers enco mpassing a 149 bp target region around codon 12 of the H-ras proto-onc ogene. Quantitative DNA damage specific response was observed with nan ogram amounts of genomic DNA. This approach allowed analysis of the in itial DNA damage at a level less than 1 anti-BPDE adduct per 6.4 kbp r as gene fragment. Repair proficient GM637 cells exposed to 2 mu M anti -BPDE showed a faster removal of the adducts from the H-ras gene segme nt than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measuremen t of the repair of diverse DNA base lesions in any genomic region of k nown sequence.