Detection in situ of active polypeptides of mammalian and T₄ DNA kinases after sodium dodecyl sulfate/polyacrylamide gel electrophoresis

Abstract

DNA kinase activity of rat liver nuclei was detected in situ after electrophoresis in sodium dodecyl sulfate/ polyacrylamide gel containing 5'-hydroxyl nicked DNA as DNA substrate. After renaturation of polypeptides, the gel was incubated with [Y-~~PIATP and Mg2+. An active polypeptide corresponding to MI 61 000 was observed as a radioactive band by autoradiography. The intensity of the band was proportional to the amount of the enzyme applied. The active band common to various tissues of rat was observed with the nuclear extracts, indicating that DNA kinase for rat tissues is composed of a single polypeptide of MI 61000. In contrast, T4 polynucleotide kinase (MI = 140000) showed an active polypeptide band corresponding to the subunit of M , 33 000. DNA kinase that phosphorylates 5'-hydroxyl ends of DNA has been isolated from T2-and T4-infected Escherichia coli cells [l], rat liver [2, 31 and calf thymus [4, 51. Although DNA kinase is presumed to be involved in DNA repair, DNA replication or both in co-operation with DNA ligase that joins juxtaposed 3'-hydroxyl and 5'-phosphoryl ends at nicks in duplex DNA, the physiological function remains unknown [l, 61. In comparison with the phage-induced polynucleotide kinases, mammalian DNA kinases show markedly different properties, such as restricted substrate specificity (inactive on RNA substrate), lower K,,, for ATP (2-4 pM) and acidic pH optimum (PH 5.5) [2-61. Mammalian DNA kinase is preferable for 5'-end labeling of DNA in the presence of RNA at a lower concentration of [ Y -~~P I A T P (unpublished data). We have previously purified DNA kinase from calf thymus, which is composed of a single polypeptide of MI 54000 [5], and DNA kinase partially purified from rat liver nuclei shows an apparent MI of 80000 by gel filtration [2, 31. Recently the corresponding enzyme from rat testis is reported to have a single 38-kDa polypeptide [7]. To solve the discrepancy in molecular properties of mammalian DNA kinase, we tried to adopt a new method, the 'activity gel' method (in situ assay of enzyme after SDS-PAGE) [8] for the detection of the active polypeptide of mammalian DNA kinase in crude and impure enzyme preparations. This new method to detect mammalian

Molecular Characterization of a Human DNA Kinase

“…Gel electrophoresis of the partially purified DNA kinase activity from HeLa cells indicated that the human enzyme is a ϳ60-kDa polypeptide, and thus has a molecular mass similar to that of the proteins isolated from rat liver and calf thymus (11,12,20). The molecular mass of the 521-amino acid polypeptide encoded by the sequenced cDNA is 57,102 Da.…”

“…The only significant discrepancy has been the molecular mass assigned to the polypeptides. Earlier reports regarding the PNK purified from rat organs indicated that the protein may be an 80-kDa homodimer composed of 40-kDa polypeptides (9, 10, 18), but PNK activity in tissue extracts detected on activity gels migrated as a 60-kDa polypeptide (20). Estimates for the size of calf thymus PNK have ranged from 56 to 70 kDa (16,17).…”

Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver

“…There have been a number of reports describing the isolation and characterization of PNKs from rat organs, principally liver and testes. The size of the rat liver protein isolated from nuclei has been variously reported as 61 and 80 kDa [Teraoka et al, 1975;Levin and Zimmerman, 1976;Ohmura et al, 1987;Habraken and Verly, 1988], and that of rat testes as 38 kDa [Bosdal and Lillehaug, 1985]. However, all the proteins appear to phosphorylate DNA specifically, labelling RNA either poorly or not at all, and have a pH optimum of ,5.5.…”

Spermidine and abscisic acid-mediated phosphorylation of a cytoplasmic protein from rice root in response to salinity stress

“…After washing with TBS [pH 7.4] several times, renaturation buffer (140 mM NaCl, 10 mM Tris-HCl [pH 7.4] containing 2 mM DTT, 2 mM EDTA, 1% BSA and 0.1% NP 40) was added and the blot incubated overnight at 4°C with gentle rocking. The blot was then treated with blocking buffer (30 mM Tris-HCl [pH 7.4], 5% BSA) followed by in situ phosphorylation according to Ohmura et al (1987). The blot was further processed as described by Dasgupta (1994).…”

“…In-gel activity assay was performed according to Ohmura et al (1987) with some modifications; 40 lg of protein (from 3 and 12 days' old root S 80 ) along with prestained protein molecular weight marker was electrophoresed in 10% SDS-PAGE that contained one of the following copolymerized protein substrates: 0.25 mg ml -1 myelin basic protein (MBP), 0.5 mg ml -1 histone or 0.5 mg ml -1 casein. After electrophoresis, SDS was removed and the protein was denatured by washing the gels three times in buffer (25 mM Tris-Cl [pH 7.5], 6 M Guanidium-HCl, 0.5 mM DTT, 0.1 mM Na 3 VO 4 , 0.5 mg ml -1 BSA, 0.1% Triton X-100 and 5 mM NaF) with gentle rocking at room temperature followed by renaturation in buffer (25 mM Tris-Cl [pH 7.5], 0.5 mM DTT, 0.1 mM Na 3 VO 4 and 5 mM NaF) at 4°C overnight with three changes.…”