Replication blocks and sequence interaction specificities in the codon 12 region of the c-Ha-ras proto-oncogene induced by four carcinogens in vitro.
We have examined the region surrounding codon 12 in the human c-Ha-ras proto-oncogene in vitro to determine the reaction intensities at the guanine nucleotides after exposure to the mycotoxin aflatoxin B1, to one of its human metabolites aflatoxin M1, to dimethyl sulfate, and to the major ultimate carcinogen of benzo(a)pyrene, benzo(a)pyrene diol-epoxide. Among the adducts produced, those at N-7-guanyl sites are alkali-labile and can be identified using a variation of the Maxam-Gilbert sequencing procedure. Data indicate that the guanine nucleotides of codon 12 have above average potential for adduct formation by the genotoxins when compared to other guanine sites, but were not the strongest sites. This codon 12 region has been inserted into single-stranded M13 phage, exposed to several of the genotoxins, and used as a template for DNA synthesis in vitro. There is a sequence-specific variation in polymerase inhibition at various adducted nucleotide sites, but replication blocks are not preferentially seen at the carcinogen-adducted guanines of codon 12. These results indicate that the predominance of point mutations which are detected in vivo at codon 12 do not reflect sequence-mediated preferential susceptibility of these sites to initial DNA adduction or to replication errors. The mechanisms controlling these sequence effects are not currently understood, and attempts to predict relative alkylation or termination frequencies based solely on local DNA sequence are not reliable.
Mariën
K
K
Department of Food Science and Technology, Oregon State University, Corvallis 97331.
Mathews
K
K
van Holde
K
K
Bailey
G
G
D016604:Aflatoxin B1; D016607:Aflatoxin M1; D000348:Aflatoxins; D001483:Base Sequence / Q000187:drug effects; D001580:Benzopyrenes; D002273:Carcinogens; D003062:Codon / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects*; D011905:Genes, ras / Q000187:drug effects*; D006801:Humans; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000187:drug effects*
D000348:Aflatoxins; D001580:Benzopyrenes; D002273:Carcinogens; D003062:Codon; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D016607:Aflatoxin M1; C039385:7,8-dihydroxy-9,10-epoxy-8-methyl-7,8,9,10-tetrahydrobenzo(a)pyrene; D016604:Aflatoxin B1