5'phosphorylation of DNA in mammalian cells: identification of a polymin P-precipitable polynucleotide kinase.
Proteins that catalyze 5' phosphorylation of an oligodeoxyribonucleotide substrate can be fractionated by polymin P treatment of whole cell extracts of calf thymus glands. Anion exchange chromatography on Q-Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, Polymin P-precipitable polynucleotide kinase (PP-PNK), was further purified with a 1,500-fold increase of specific activity compared to the crude Polymin P pellet fraction. Oligonucleotides, a dephosphorylated 2.9-kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3' monophosphate was not a substrate. PP-PNK preparations exhibited an apparent KM of 52 microM for ATP and 8 microM for oligo dT25. The enzyme preparation displayed no detectable 3' phosphatase or cyclic 2',3' phosphohydrolase activities. The sedimentation coefficient of the PP-PNK activity was 3.8S as determined by sucrose density gradient analysis; the Stokes radius was 45 A, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimum in the neutral to alkaline range in several buffer systems and is distinct from the DNA kinase with an acidic pH optimum previously described in calf thymus.
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Department of Medicine, McGill University, Montreal, Quebec, Canada.
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D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010766:Phosphorylation; D011094:Polyethyleneimine / Q000737:chemistry*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry / Q000302:isolation & purification*; D012492:Salts / Q000737:chemistry; D013950:Thymus Gland / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D012492:Salts; C027903:oligo (dT); D011094:Polyethyleneimine; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase