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Enzymatic conversion of deoxycytidine 5′-monophosphate to 5-methyldeoxycytidine 5′-triphosphate

✍ Scribed by Richard Y.-H. Wang; Lan-Hsiang Huang; Melanie Ehrlich

Publisher
Elsevier Science
Year
1982
Tongue
English
Weight
465 KB
Volume
122
Category
Article
ISSN
0003-2697

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✦ Synopsis

An enzymatic method has been developed to synthesize S-methyldeoxycytidine triphosphate (m'dCTP) starting with dCMP and formaldehyde as substrates. Methylation is catalyzed by a dCMP methyltransferase in the presence of tetrahydrofolic acid. The resulting m'dCMP is converted to m'dCTP by a kinase preparation and an ATP-regenerating system. The methyltransferase and kinase preparations were isolated from extracts of bacteriophage XP-12infected Xanrhomonas oryzae by a single-step chromatography on DEAE-cellulose. About 10% of the input radioactivity from 14C-or 'H-labeled formaldehyde was recovered in m'dCTP under our reaction conditions. Using this enzymatically synthesized m5dCTP, up to 60% of the cytosine residues in DNA was replaced by 5-methylcytosine residues in a nick translation reaction catalyzed by Escherichia coli DNA polymerase 1.

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