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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.

The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 "C and using the doublestranded DNA substrate (dA), . [(dT),,], ilo. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true K , for the DNA substrate was 0.6 pM and that of ATP 100 pM.Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32P-The joining reaction was inhibited by high concentrations, i.e. above approximately 70 mM, of salts such as KC1, NaCl, NH,Cl and CsC1. At a concentration of 200 mM almost 100 inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent K,,, for the DNA substrate whereas the apparent K,,, for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.as substrate. No breakdown of this DNA could be detected.Polynucleotide ligases have now been isolated from a variety of organisms [l]. The enzymes studied most thoroughly appear to be those obtained from uninfected and from T4-infected Escherichia coli. Both enzymes have been isolated in a homogeneous state and been shown to consist of a single peptide chain of similar molecular weight [2,3]. Important differences have, however, been detected between the enzymes. Thus the T4 enzyme requires ATP as a cofactor while the enzyme from E. coli employs NAD. Furthermore, T4 polynucleotide ligase, in contrast to the enzyme from uninfected E. coli, is able to catalyze the joining of oligoribonucleotides when annealed to a polydeoxyribonucleotide strand as well as that of oligodeoxyribonucleotides when annealed to a polyribonucleotide strand [4,5]. Also, Ahbreviu~ions. The abbreviations used for nucleotides and polynucleotides are as recommended by TUPAC-IUB Commission, see Eur. .I. Biochem. IS, 203-208 (1970). (dA), = poly(deoxyadeny1ic acid); (dA-dT), = alternating deoxyadenylate and thymidylate chains; (dT),, = oligo(thymidy1ic acid) containing 10 monomers and having a phosphate group at the 5' end; (dA), . [(dT)lo]n;lo = a double-stranded DNA containing poly(deoxyadeny1ic acid) in one strand and oligomers of (dT),, in the other, the amount of monomer in each strand being the same.

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