Interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with oligonucleotides containing native or modified (defective) recognition sites.
The DNA-[N 6-adenine]-methyltransferase (Dam MTase) of phage T4 catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have used a gel shift assay to monitor complex formation between T4 Dam and various synthetic duplex oligonucleotides, either native or modified/defective. The results are summarized as follows. (i) T4 Dam bound with approximately 100-fold higher affinity to a 20mer specific (GATC-containing) duplex containing the canonical palindromic methylation sequence, GATC, than to a non-specific duplex containing another palindrome, GTAC. (ii) Compared with the unmethylated duplex, the hemimethylated 20mer specific duplex had a slightly increased ( approximately 2-fold) ability to form complexes with T4 Dam. (iii) No stable complex was formed with a synthetic 12mer specific (GATC-containing) duplex, although T4 Dam can methylate it. This indicates that there is no relation between formation of a catalytically competent 12mer-Dam complex and one stable to gel electrophoresis. (iv) Formation of a stable complex did not require that both strands be contiguous or completely complementary. Absence of a single internucleotide phosphate strongly reduced complex formation only when missing between the T and C residues. This suggests that if T4 Dam makes critical contact(s) with a backbone phosphate(s), then the one between T and C is the only likely candidate. Having only one half of the recognition site intact on one strand was sufficient for stable complex formation provided that the 5'G.C base-pairs be present at both ends of the palindromic, GATC. Since absence of either a G or C abolished T4 Dam binding, we conclude that both strands are recognized by T4 Dam.
Malygin
E G
EG
Institute of Molecular Biology, State Research Center of Virology, and Biotechnology 'Vector', Koltsovo, Novosibirsk Region 633159, Russia and Department of Biology, University of Rochester, Rochester, NY 14627-0211, USA.
Petrov
N A
NA
Gorbunov
Y A
YA
Kossykh
V G
VG
Hattman
S
S
D017122:Bacteriophage T4 / Q000201:enzymology*; D019175:DNA Methylation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D012436:S-Adenosylmethionine; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D012436:S-Adenosylmethionine; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499297:dam protein, Bacteriophage T4