Bacteriophage T4 Dam DNA-(N6-adenine)-methyltransferase. Processivity and orientation to the methylation target.
We carried out steady state and pre-steady state (burst) kinetic analyses of the bacteriophage T4 Dam DNA-(N(6)-adenine)-methyltransferase (MTase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to Ade in oligonucleotide duplexes containing one or two specific GATC sites with different combinations of methylated and unmodified targets. We compared the results for ligated 40-mer duplexes with those of the mixtures of the two unligated duplexes used to generate the 40-mers. The salient results are as follows: (i) T4 Dam MTase modifies 40-mer duplexes in a processive fashion. (ii) During processive movement, T4 Dam rapidly exchanges product S-adenosyl-l-homocysteine (AdoHcy) for substrate AdoMet without dissociating from the DNA duplex. (iii) T4 Dam processivity is consistent with an ordered bi-bi mechanism AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. However, in contrast to the steady state, here DNA(Me) upward arrow signifies departure from a methylated site GMTC upward arrow without physically dissociating from the DNA. (iv) Following methyl transfer at one site and linear diffusion to a hemimethylated site, a reconstituted T4 Dam-AdoMet complex rapidly reorients itself to the (productive) unmethylated strand. T4 Dam-AdoHcy cannot reorient at an enzymatically created GMTC site. (v) The inhibition potential of fully methylated sites 5'-GMTC/5'-GMTC is much lower for a long DNA molecule compared with short single-site duplexes.
Zinoviev
Victor V
VV
Institute of Molecular Biology, State Research Center of Virology and Biotechnology Vector, Novosibirsk 630559, Russia.
Evdokimov
Alexey A
AA
Malygin
Ernst G
EG
Schlagman
Samuel L
SL
Hattman
Stanley
S
D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D014764:Viral Proteins
D017931:DNA Primers; D014764:Viral Proteins; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499297:dam protein, Bacteriophage T4