Phosphorylation of double-stranded DNAs by T₄ polynucleotide kinase

Abstract

The phosphorylation by T4 polynucleotide kinase of various double-stranded DNAs containing defined 5'-hydroxyl end group structures has been studied. Particular emphasis was placed on finding conditions that allow complete phosphorylation. The DNAs employed were homodeoxyoligonucleotides annealed on the corresponding homopolymers, DNA duplexes corresponding to parts of the genes for alanine yeast tRNA, and a suppressor tyrosine tRNA from Escherichia coli. The rate of phosphoylation of DNAs with 5'-hydroxyl groups in gaps was approximately ten times slower than for the corresponding single-stranded DNA. At low concentrations of ATP, 1 muM, incomplete phosphorylation was obtained, whereas with higher concentrations of ATP, 30 muM, complete phosphorylation was achieved. In the case of DNAs with 5'-hydroxyl groups at nicks approximately 30% phosphorylation could be detected using 30 muM ATP. A DNA containing protruding 5'-hydroxyl group ends was phosphorylated to completion using the same conditions as for single-stranded DNA, i.e., a ratio between the concentrations of ATP and 5'-hydroxyl groups of 5:1 and a concentration of ATP of approximately 1 muM. For a number of DNAs containing protruding 3'-hydroxyl group ends and one DNA containing even ends incomplete phosphorylation was found under similar conditions. For all these DNAs a plateau level was observed varying from 20 to 45% of complete phosphorylation. At 20 muM and higher ATP concentrations, the phosphorylation was complete also for these DNAs. With low concentrations of ATP a rapid production of inorganic phosphate was noted for all the latter DNAs. The apparent equilibrium constants for the forward and reverse reaction were determined for a number of different DNAs, and these data revealed that the plateau levels of phosphorylation obtained at low concentrations of ATP for DNAs with protruding 3'-hydroxyl group and even ends is not a true equilibrium resulting from the forward and reverse reaction. It is suggested that the plateau levels are due to formation of inactive enzyme-substrate and enzyme-product complexes. For all double-stranded DNAs tested, except DNAs containing protruding 5'-hydroxyl group ends, addition of KCl to the reaction mixture resulted in a drastic decrease in the rate of phosphorylation, as well as in the maximum level phosphorylated. Spermine, on the other hand, had little influence. Both of these agents have previously been shown to activate T4 polynucleotide kinase using single-stranded DNAs as substrates (Lillehaug, J.R., and Kleppe, K. (1975), Biochemistry 14, 1221). The inhibition of phosphorylation of double-stranded DNAs by salt might be the result of stabilization of the 5'-hydroxyl group regions of these DNAs.

Characterization of Polynucleotide Kinase/Phosphatase Enzymes from Mycobacteriophages Omega and Cjw1 and Vibriophage KVP40

“…In agreement with Lillehaug et al (43), we found that T4 Pnkp preferentially labeled the 5Ј single-stranded BamHI overhang when the reactions were performed at 1 M ATP (Fig. 8, middle), and that the end specificity was relaxed when the ATP concentration was increased to 50 M. This contrasts with Omega Pnkp, which labeled 5Ј-OH termini at 5Ј overhangs, 3Ј overhangs, and blunt ends with similar efficiencies at either 1 or 50 M ATP.…”

Regulation of lipoprotein lipase in the diabetic rat.

“…LPL mRNA was visualized by hybridization using a 39-nucleotide oligomer that was synthesized (courtesy of Dr. J. Eberwine, University of Pennsylvania) to be complementary to the region of mouse LPL mRNA encoding for amino acids 15-27. The probe was prepared by end-labeling using T4 polynucleotide kinase (13) and gives a signal comparable to that obtained using a rat LPL cDNA (9). a-Actin cDNA (a kind gift of Dr. L. Kedes, University ofSouthern California) was nick-translated according to standard protocols (14).…”

Structural analysis of the sequence coding for an inducible 26‐kDa protein in human fibroblasts

“…l), were labelled externally at their 5' ends by kination [14] on a dephosphorylated restriction digest of the appropriate total cDNA (i.e. 26K-2 or 26K-7).…”

“…determination of the cDNA inserts or of the genomic DNA segments was performed following the chemical modification and degradation procedure of Maxam and Gilbert [15]. Appropriate fragments were end-labelled, either by dephosphorylation and kination [14], or by filling-in at the 3' end with Klenow polymerase [16].…”