Pre-steady state kinetics of bacteriophage T4 dam DNA-[N(6)-adenine] methyltransferase: interaction with native (GATC) or modified sites

• Published in: Nucleic acids research 2000-11, Vol.28 (21), p.4207-4211
• Main Authors: Malygin, E G, Lindstrom, Jr, W M, Schlagman, S L, Hattman, S, Reich, N O
• Format: Article
• Language: English
• Subjects: Bacteriophage T4 - enzymology; Base Sequence; Binding Sites; Catalysis; DNA adenine-(N6) methyltransferase; DNA Methylation; DNA methyltransferase; DNA-Binding Proteins - metabolism; Kinetics; Mutation - genetics; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - genetics; Oligodeoxyribonucleotides - metabolism; Phage T4; Protein Binding; Regulatory Sequences, Nucleic Acid - genetics; S-adenosylmethionine; S-Adenosylmethionine - metabolism; Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism; Substrate Specificity; Viral Proteins
• ISSN: 1362-4962; 0305-1048; 1362-4962
• DOI: 10.1093/nar/28.21.4207

The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S:-adenosyl-L-methionine (AdoMet) to the N(6)-position of adenine [generating N(6)-methyladenine and S:-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant k(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s(-1), respectively) was at least 20-fold larger than the overall reaction rate constant k(cat) (0.023 s(-1)). This indicates that the release of products is the rate-limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit k(meth). Preformed T4 Dam MTase-DNA complexes are less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.