Kinetics and Effect of Salts and Polyamines on T4 Polynucleotide Ligase

Abstract

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.

Kinetic Mechanism of the Mg2+-dependent Nucleotidyl Transfer Catalyzed by T4 DNA and RNA Ligases

“…In the past, the pyrophosphate exchange reaction catalyzed by these enzymes have been studied, and the apparent K m values for ATP in the DNA-joining reaction on different DNA substrates have been determined (4,5,8,9). X-ray structures of the related enzymes, T7 DNA ligase and mRNA capping enzyme from Chlorella virus PBCV-1 in complex with the nucleoside triphosphate as well as the enzyme-adenylylate complex of DNA ligase from Chlorella virus PBCV-1 have been solved (10 -12).…”

T4 polynucleotide ligase catalyzed joining on triple-stranded nucleic acids

“…In the present work, the action of Tq polynucleotide ligase on synthetic triple-stranded DNAs has been investigated. In a previous publication from this laboratory, the mechanism of action of T4 polynucleotide ligase on synthetic double-stranded DNA has been described in detail (Raae et al, 1975a). The results of the present work suggest that this enzyme can also carry out joining of phosphodiester linkages on some triplestranded nucleic acids.…”

“…W e have previously shown that low concentrations of the polyamines putrescine, cadaverine, and TiME ( m i d spermidine (up to 1 mM) give a small activation of T4 polynucleotide ligase catalyzed joining of dTlo in a double-stranded substrate (Raae et al, 1975a). A t higher concentrations, however, a marked inhibition was obtained.…”

“…Using the equation obtained for native double-stranded DNA, relating sedimentation coefficient to molecular weight, ~2 0 ,~ = 0.0882M0.346 (Studier, 1965), a molecular weight of 1.08 X lo6 was obtained for the peak fraction of the 1 :I complex. Assuming that the same equation also is valid for a triple-stranded DNA, the molecular weight of the peak fraction a : 0 2 a The analvses were carried out essentiallv as described bv Raae et al (1975a).…”

Ionic regulation in genetic translation systems.

“…These occur not only under the influence of polyamines or polyanions such as heparin (9,11,12), but also when the reactions are subjected to promoters, repressors, and other physiologically important factors (7,13,14). One can postulate that most of the protein factors behave as polyelectrolytes, promoting and suffering from modifications of the local electrostatic potential and therefore of the local concentration of protons, pKs(4,) and, finally, catalytic activity.…”