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Nucleic Acids Research, 1989, Vol. 17, No. 21 8475-8483
© 1989

MOLECULAR BIOLOGY

Purification of the E.coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethyiguanine and O4-methylthymine in dodecadeoxyribnucleotides

M.C. Wilkinson1,2, P.M. Potter1, L. Cawkwell1, P. Georgiadis3, D. Patel3, P.F. Swann3 and G.P. Margison*

1Department of Carcinogencsis, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute Manchester M20 9BX, UK 2School of Biological Sciences, University College of North Wales Bangor, Gwynedd LL57 2UW, UK 3Biochemistry Department, University College and Middlesex School of Medicine, Middlesex Hospital London W1P 7PN, UK

*To whom correspondence should be addressed

Received August 22, 1989. Revised September 22, 1989. Accepted September 22, 1989.

The E.coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme ({small tilde} 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and (O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG > O6-EtG > O4-MeT, however, the ogt protein was found to repair O6MeG, O6-EtG and (O4-MeT, 1.1, 173 and 84 times, respectively, faster than the ada protein.


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