A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase.
The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.
Malygin
E G
EG
Institute of Molecular Biology, State Research Center of Virology and Biotechnology 'Vector', Novosibirsk 633159, Russia.
Evdokimov
A A
AA
Zinoviev
V V
VV
Ovechkina
L G
LG
Lindstrom
W M
WM
Reich
N O
NO
Schlagman
S L
SL
Hattman
S
S
D015075:2-Aminopurine / Q000737:chemistry / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D005453:Fluorescence; D005976:Glutaral / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012435:S-Adenosylhomocysteine / Q000378:metabolism / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000378:metabolism* / Q000502:physiology; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013997:Time Factors; D014764:Viral Proteins
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D014764:Viral Proteins; D015075:2-Aminopurine; D012436:S-Adenosylmethionine; D004247:DNA; D012435:S-Adenosylhomocysteine; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499297:dam protein, Bacteriophage T4; D005976:Glutaral