Covalent labelling of the Klenow fragment of DNA-polymerase I from E. coli

Incubation of the Klenow fragment of E. coli DNA polymerase I with [alpha-32P] dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such as beta-mercaptoethylamine, efficiently inhibiting the labell...

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Bibliographic Details
Published in:	Bioorganicheskaia khimiia Vol. 15; no. 10; p. 1356
Main Authors:	Degtiarev, S Kh, Zaĭchikov, E F, Lavruk, O I, Mitina, R L, Mustaev, A A, Rikhter, V A
Format:	Journal Article
Language:	Russian
Published:	Russia (Federation) 01-10-1989
Subjects:	Chromatography, High Pressure Liquid DNA Polymerase I - metabolism DNA-Directed DNA Polymerase - metabolism Electrophoresis, Polyacrylamide Gel Escherichia coli - enzymology Hydrogen-Ion Concentration Nucleotides - metabolism Substrate Specificity
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Summary:	Incubation of the Klenow fragment of E. coli DNA polymerase I with [alpha-32P] dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such as beta-mercaptoethylamine, efficiently inhibiting the labelling. It is suggested that radiolabelling of the enzyme is the result of formation of chemically active products of the radiolysis of [alpha-32P]NTP (which are likely to be radicals). Non-radioactive NTP hinder the labelling, whereas Mg2+ and polynucleotide do not affect it. Cleavage of the enzyme by hydroxylamine and cyanogen bromide and analysis of gel-electrophoretic patterns of the cleavage products led to conclusion that 32P-label is located between Gly-544 and Met-647.
ISSN:	0132-3423