Construction and characterization of extrachromosomal probes for mutagenesis by carcinogens: site-specific incorporation of O6-methylguanine into viral and plasmid genomes.

Abstract

Organic synthesis and recombinant DNA technology were used to situate a putatively premutagenic DNA lesion, O6-methylguanine (O6MeGua), at a specific location in the genomes of 2 bacterial viruses, M13mp8 and .vphi.X174, and of the bacterial plasmid pBR322. In each genome the 1st guanine residue in the unique recognition sequence for restriction endonuclease Pst I (5''-C-T-G-C-A-G-3'') was replaced with O6MeGua. This was accomplished by ligating a chemically synthesized tetranucleotide, 5''-pTpm6GpCpA-3'', into a circular, genome-length heteroduplex in which the 4 internal nucleotides of the Pst I recognition site were removed from 1 strand of the DNA double helix (ligation yield, .apprxeq. 50%). The tetranucleotide was located specifically at the Pst I site and the presence of O6MeGua rendered the ligation product resistant to cleavage by Pst I. Sensitivity of the genome to Pst I was restored upon treatment with purified Escherichia coli O6MeGua DNA-methyltransferase, a repair protein that removes the methyl group from DNA-bound O6MeGua. This result, in combination with other data, showed unambiguously that O6MeGua was incorporated with high yield into the Pst I recognition sequence.