CYTOSINE METHYLATION DETERMINES HOT-SPOTS OF DNA-DAMAGE IN THE HUMAN P53 GENE

In the P53 tumor suppressor gene, a remarkably large number of somatic mutations are found at methylated CpG dinucleotides. We have previous ly mapped the distribution of (+/-) anti-7 beta,8 alpha-dihydroxy-9 al pha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) adducts al ong the human P53 gene [Denissenko, M. F., Pao, A., Tang, M.-s. & Pfei fer, G. P. (1996) Science 274, 430-432]. Strong and selective formatio n of adducts occurred at guanines in CpG sequences of codons 157, 248, and 273, which are the major mutational hot spots in lung cancer. Chr omatin structure was not involved in preferential modification of thes e sites by BPDE. To investigate other possible mechanisms underlying t he selectivity of BPDE binding, we have mapped the adducts in plasmid DNA containing genomic P53 sequences. The adduct profile obtained was different from that in genomic DNA. However, when cytosines at CpG seq uences were converted to 5-methylcytosines by the CpG-specific methyla se SssI and the DNA was subsequently treated with BPDE, adduct hot spo ts were created which were similar to those seen in genomic DNA where all CpGs are methylated. A strong positive effect of 5-methylcytosine on BPDE adduct formation at CpG sites was also documented with sequenc es of the PGK1 gene derived from an active or inactive human X chromos ome and having differential methylation patterns. These results show t hat methylated CpG dinucleotides, in addition to being an endogenous p romutagenic factor, may represent a preferential target for exogenous chemical carcinogens. The data open new avenues concerning the reasons that the majority of mutational hot spots in human genes are at CpGs.