A DNA Methylase from Thermus thermophilus HB8

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Abstract
A DNA methylase was purified in a homogeneous state from a extremely thermophilic bacterium, Thermus thermophilus HB8, by chromatography on, successively, phosphocellulose, CM-cellulose, and heparin-Sepharose. The molecular weight of the enzyme was determined to be about 44,000 by gel filtration on a Sephadex G-100 column and 41,000 by SDS-poIy-acrylamide gel electrophoresis, and these findings suggest a single polypeptide enzyme. The enzyme develops maximum activity around pH 7.4 and at 70°C. Enzymatic activity is completely inhibited by 0.2M NaCl or 2 mM HgCI2. The enzyme transfers methyl groups from S-adenosyl-L-methionine to a double stranded DNA. The sole product of the reaction was identified as N-6-methyl adenine after hydrolysis of the DNA with formic acid. The enzyme kinetics obey the Michaelis-Menten equation and Km values for S-adenosylmethionine and lambda phage DNA were determined to be 0.8 μm and 10 μg/ml, respectively. The enzyme does not transfer methyl groups to 7thHB8I endonuclease digested DNA as well as the host (T. thermophilus HB8) DNA. The number of methyl groups of the fully methylated ø×174 RF DNA was about twice as many as TthHB8I endonuclease sites on the DNA. The distribution of the methyl groups of ø×174 RF DNA among the HaeIII fragments was the same as that of TthHB81 endonuclease sites, suggesting that this DNA methylase is the other component of the modification-restriction system including TthHB8I endonuclease. The enzyme probably recognizes the sequence, 5′-TCGA-3′, in a double stranded DNA and probably methylates adenine in the above sequence.