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.

The structural basis for the mutagenicity of O 6 -methyl-guanine lesions

“…The cytotoxic effects of DNA-methylating agents have been exploited in their use as potent anticancer agents. O 6 -methyl-guanine (O6MeG) is mutagenic because polymerases frequently misinsert T opposite O6MeG instead of C, both in vivo (9,10) and in vitro (11)(12)(13). In this study, we present the crystal structures of complexes of a high-fidelity DNA polymerase with substrates representing several steps of nucleotide insertion opposite O6MeG.…”

Extrachromosomal Probes for Mutagenesis by Carcinogens: Studies on the Mutagenic Activity of O 6 -Methylguanine Built into a Unique Site in a Viral Genome

“…This high yield indicated that the tetramer, when present in large excess, was sufficiently complementary to the ligation target for DNA ligase to very effectively form phosphodiester bonds. Electrophoresis followed by autoradiography of the ligation mixture revealed that incorporation of [32p] occurred principally in the circular heteroduplexes (22). The heteroduplex DNA structures that received the modified oligonucleotide had gaps that formed with equal probability in either of the two complementary strands (Fig.…”

“…The first step in this procedure (22) was to treat the replicative form (RF) of M13mp8 with Pst I (Fig. 2).…”

“…The adduct-containing genomes shown as ligation products at the bottom of Figure 2B were subjected to a series of characterization experiments (22). One of these experiments established that both ends of the modified tetranucleotide had ligated into the gap originally present in the duplex genome; it was important that this step went to completion since failure of one of the ends to ligate would have left a nick in the DNA molecule near the adduct, and such a nick could have provided a mechanism for the efficient repair of the adducted genome upon its entry into cells.…”

A chemical perspective on the anthracycline antitumor antibiotics.

“…These nucleophiles are, however, limited to thiols, and do not include more relevant nucleophiles such as enols or phosphate esters (such as GMP). Although negative results with GMP may reflect on the possibility of covalent DNA modification, it must be remembered that mononucleotides significantly underestimate the reactivity of oligonucleotides as nucleophiles (19,20).…”