Dam methyltransferase from Escherichia coli: kinetic studies using modified DNA oligomers: nonmethylated substrates.
Steady-state kinetics of the N6-adenine Dam methyltransferase have been measured using as substrates non-self-complementary tetradecanucleotide duplexes that contain the GATC target sequence. Modifications in the GATC target sequence of one or both of the strands included substitution of guanine by hypoxanthine, thymine by uracil or 5-ethyl-uracil and adenine by diamino-purine (2-amino-adenine). Thermodynamic parameters for the 14-mer duplexes were also determined. DNA methylation of duplexes containing single dl for dG substitution of the Dam recognition site was little perturbed compared with the canonical substrate. Replacement of dG residues by dl in both strands resulted in a decrease of the specificity constant. Substitution in both strands appears to be cumulative. Substitution of the methyl-accepting adenine residues by 2-amino-adenine resulted in surprisingly little perturbation. Dam methyltransferase is rather tolerant to different substitutions. The results show much less spread than those for the analogous hemimethylated substrates studied previously (Marzabal et al., 1995). The absence of the methylation marker appears to be deleterious to the specificity of the transition state of the active complex, while the binding of the DNA substrate to the enzyme appears to be mostly determined by the thermodynamic stability of the DNA duplex.
Thielking
V
V
CEA/Saclay, Service de Biochimie et Génétique Moléculaire, Gif-sur-Yvette, France.
Dubois
S
S
Eritja
R
R
Guschlbauer
W
W
D015075:2-Aminopurine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000225:Adenine / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D019175:DNA Methylation; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006147:Guanine / Q000737:chemistry / Q000378:metabolism; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D015345:Oligonucleotide Probes; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012984:Software; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry / Q000378:metabolism; D014498:Uracil / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
D015342:DNA Probes; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D015345:Oligonucleotide Probes; D019271:Hypoxanthine; C048011:5-ethyluracil; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D014498:Uracil; D006147:Guanine; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499294:dam protein, E coli; D000225:Adenine; D013941:Thymine