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 two bacterial viruses, M13mp8 and phi X174, and of the bacterial plasmid pBR322. In each genome the first 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 four internal nucleotides of the Pst I recognition site had been removed from one strand of the DNA double helix (ligation yield, approximately equal to 50%). It was established that the tetranucleotide was located specifically at the Pst I site and that 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.