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Journal of Biosciences

, Volume 11, Issue 1–4, pp 215–224 | Cite as

Purification and some properties of human DNA-O6-methylguanine methyltransferase

  • Amy M. Boulden
  • Robert S. Foote
  • G. Scott Fleming
  • Sankar Mitra
Article

Abstract

DNA-O6-methylguanine methyltransferase was purified from the nuclear fraction of fresh human placenta using ammonium sulphate precipitation, gel filtration, affinity chromatography on DNA-cellulose and hydroxyapatite. The methyltransferase preparation was approximately 1–2% pure based on specific activity, and was free of nucleic acids. The protein reacts stoichiometrically with O6-methylguanine in DNA with apparent second-order kinetics. The human methyltransferase has a pH optimum of about 8.5, similar to that of the corresponding rat and mouse proteins. NaCl inhibits the reaction in a concentration-dependent fashion. The human protein, like the rodent andE. coli methyltransferases, needs no cofactor. While lmM MnCl2, lmM spermidine, 5mM MgCl2 and 10 mM EDTA individually do not significantly inhibit the initial rate of reaction, the protein is nearly completely inactive in 5 mM A1Cl3 or FeCl2 or 10 mM spermidine. The initial rate of reaction increases as a function of temperature at least up to 42°. The reaction is inhibited by DNA in a concentration-dependent manner, with single-stranded DNA being more inhibitory than duplex DNA.

Keywords

DNA repair DNA alkylation O6-methylguanine human methyltransferase 

Abbreviations used

EDTA

ethylenediaminetetraacetic acid

DTT

dithiothreitol

αToSF

α-toluenesulphonyl fluoride

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Copyright information

© Indian Academy of Sciences 1987

Authors and Affiliations

  • Amy M. Boulden
    • 1
  • Robert S. Foote
    • 1
  • G. Scott Fleming
    • 1
  • Sankar Mitra
    • 1
    • 2
  1. 1.Oak Ridge National LaboratoryThe University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences Biology DivisionOak RidgeUSA
  2. 2.Biology DivisionOak Ridge National LaboratoryOak RidgeUSA

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