The DNA [adenine-N6]methyltransferase (Dam) of bacteriophage T4.
A functional bacteriophage T4 dam+ gene, which specifies a DNA [adenine-N6]methyltransferase (Dam), was cloned on a 1.8-kb HindIII fragment [Schlagman and Hattman, Gene 22 (1983) 139-156]. Sequence analysis [Macdonald and Mosig, EMBO J. 3 (1984) 2863-2871] revealed two overlapping in-phase open reading frames (ORFs). The 5' proximal ORF initiates translation at an AUG and encodes a 30-kDa polypeptide, whereas the downstream ORF initiates translation at a GUG and encodes a 26-kDa polypeptide. Analysis of BAL 31 deletions in our original dam+ clone has verified that at least one of these overlapping ORFs, in fact, encodes T4 Dam. To investigate where T4 Dam translation is initiated, we have constructed plasmids in which a tac or lambda PL promoter is placed 5' to either the longer ORF or just the shorter ORF. Only clones which contain a promoter in front of the longer ORF produce active T4 Dam. This indicates that the 26-kDa polypeptide alone cannot be T4 Dam. Additional experiments suggest that only the 30-kDa polypeptide is required for enzyme activity and that the shorter ORF is not translated in plasmid-carrying cells. We also present evidence that T4 Dam is capable of methylating 5'-GATC-3', GATm5C, and GAThmC sequences; non-canonical sites (e.g., GACC) are also methylated, but much less efficiently.
Schlagman
S L
SL
Department of Biology, University of Rochester, NY 14627.
Miner
Z
Z
Fehér
Z
Z
Hattman
S
S
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)