Physicochemical properties of T4 polynucleotide kinase.

Some physicochemical properties of T4 polynucleotide kinase (EC2.7.1.78) have been studied. The enzyme is an oligomer of one polypeptide chain. The molecular weight of the monomer is 33000, as determined from the amino acid analysis. Phenylalanine is the N-terminal amino acid. Each monomer contains two --SH groups, one exposed and one more buried. Circular dichroic spectra suggest a high content of alpha-helical structure, 45--55%. Excitation at 280 nm gave a strong emission fluorescence spectrum with a maximum centering at 340 nm. Sedimentation studies suggested the enzymically active form to be a tetramer. High ionic strength (0.1 M KC1), spermine, and the substrates ATP and thymidine 3'-monophosphate were found to be essential factors in order to stabilize the protein in an oligomeric structure. The association constants for ATP, thymidine 3'-monphosphate, and P1 were determined fluorimetrically to be 7.9 x 105, 4.8 x 105, and 7.2 x 10(2) M-1 respectively.     

3'-Phosphatase activity in T4 polynucleotide kinase.

The purification of T4 polynucleotide kinase results in the copurification of an activity which will specifically remove the 3'-terminal phosphate from a variety of deoxyribonucleotides and ribonucleotides in the absence of ATP. This phosphatase activity requires magnesium, has a pH optiumum of 6.0, and is more active with deoxyribonucleotides than ribonucleotides. T4 polynucleotide kinase and the 3'-phosphatase activity copurify by gradient elution column chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite. The two activities are included in and comigrate on Sephadex G-200. Polyacrylamide gel electrophoresis at PH 9.2 results in conigration of the two activities together with the major protein band. The two activities respond in parallel to heat inactivation at 35 degrees C and ATP, a substrate for the kinase only, protects both activities from heat inactivation. It is therefore suggested that the two activities are functions of the same protein molecule.     

Stabilization of T4 polynucleotide kinase by macromolecular crowding.

T4 polynucleotide kinase rapidly loses activity during its reaction on duplex DNA termini. Addition of high concentrations of nonspecific polymers reverses or prevents this inactivation. In contrast, additions of related materials of lower molecular weight are relatively ineffective in stabilizing the kinase. Such a pattern suggests that the stabilizing effects of polymers on kinase activity are due to macromolecular crowding. An effect of crowding on the known tendency of the kinase to undergo oligomerization reactions is consistent with our observations.     

Effect of salts and polyamines on T4 polynucleotide kinase.

The activity of T4 polynucleotide kinase (EC 2.7.1.78) was found to be greatly stimulated by salts, such as NaCl and KCl, and polyamines such as spermine and spermidine. Up to a sixfold increase in initial rates was observed with a variety of different single-stranded DNAs and mono- and oligonucleotides. The optimal concentrations of salts were 0.125 M, corresponding to a total ionic strength of mu equals 0.19. For polyamines the optimal concentrations were found to be at approximately 2 mM. With low enzyme concentration and in the absence of activators complete phosphorylation was not achieved for a number of substrates. In the presence of salts or polyamines or high concentration of enzyme the phosphorylation proceeded to completion. Addition of salt led to an increase in both the apparent V-max and the Michaelis constant for the DNA substrate whereas the Michaelis constant of ATP remained unchanged. Polyamines had a similar influence on the kinetic constants for the DNA substrate whereas a decrease was found for the apparent Michaelis constant for ATP. The overall mechanism in the presence of activators was found to be sequential but probably of a rapid equilibrium random type. Of the inorganic anions tested both P-i and PP-i inhibited the enzyme in a competitive manner with both substrates.     

Domain structure and mutational analysis of T4 polynucleotide kinase

T4 polynucleotide kinase (Pnk) is the founding member of a family of 5'-kinase/3'-phosphatase enzymes that heal broken termini in RNA or DNA by converting 3'-PO(4)/5'-OH ends into 3'-OH/5'-PO(4) ends, which are then suitable for sealing by RNA or DNA ligases. Here we employed site-directed mutagenesis and biochemical methods to dissect the domain structure of the homotetrameric T4 Pnk protein and to localize essential constituents of the apparently separate active sites for the 5'-kinase and 3'-phosphatase activities. We characterized deletion mutants Pnk(42-301) and Pnk(1-181), which correspond to domains defined by proteolysis with chymotrypsin. Pnk(1-181) is a monomer with no 3'-phosphatase and low residual 5'-kinase activity. Pnk(42-301) is a dimer with no 5'-kinase and low residual 3'-phosphatase activity. Four classes of missense mutational effects were observed. (i) Mutations K15A, S16A, and D35A inactivated the 5'-kinase but did not affect the 3'-phosphatase or the tetrameric quaternary structure of T4 Pnk. 5'-kinase activity was ablated by the conservative mutations K15R, K15Q, and D35N; however, kinase activity was restored by the S16T change. (ii) Mutation D167A inactivated the 3'-phosphatase without affecting the 5'-kinase or tetramerization. (iii) Mutation D85A caused a severe decrement in 5'-kinase activity and only a modest effect on the 3'-phosphatase; the nearby N87A mutation resulted in a significantly reduced 3'-phosphatase activity and slightly reduced 5'-kinase activity. D85A and N87A both affected the quaternary structure, resulting in a mixed population of tetramer and dimer species. (iv) Alanine mutations at 11 other conserved positions had no significant effect on either 5'-kinase or 3'-phosphatase activity.     

Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase

T4 phage polynucleotide kinase (PNK) displays 5′-hydroxyl kinase, 3′-phosphatase and 2′,3′-cyclic phosphodiesterase activities. The enzyme phosphorylates the 5′ hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonucleotide substrates of various lengths and sequences. In these structures, the 5′ ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases. The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5′-GT DNA end than for a 5′-TG end. The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique substrate molecule.     

Independent locations of kinase and 3'-phosphatase activities on T4 polynucleotide kinase

We have used two chemical modification reagents and three proteases to study the relationship between the two activities of T4 polynucleotide kinase. In each case, conditions were found where one of the two activities of the enzyme could be eliminated without greatly reducing the other. Taken together, these data indicate that the two activities are catalyzed by amino acid residues located in separate active sites on the polypeptide chain. Specific exopeptidase digestion indicates that the kinase activity lies in the NH2-terminal and the phosphatase in the COOH-terminal portion of the polypeptide chain. Partial trypsin digestion produces a 29,000-dalton fragment with no kinase activity and nearly normal 3'-phosphatase activity.     

Ferrocene-functionalized SWCNT for electrochemical detection of T4 polynucleotide kinase activity

A novel electrochemical strategy for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK) is developed by use of titanium ion (Ti(4+)) mediated signal transition coupled with signal amplification of single wall carbon nanotubes (SWCNTs). In this method, a DNA containing 5'-hydroxyl group is self-assembled onto the gold electrode and used as substrate for PNK. The biofunctionalized SWCNTs with anchor DNA and ferrocene are chosen as the signal indicator by virtue of the intrinsic 5'-phosphate end of anchor DNA and the high loading of ferrocene for electrochemical signal generation and amplification. The 5'-hydroxyl group of the substrate DNA on the electrode is phosphorylated by T4 PNK in the presence of ATP, and the resulting 5'-phosphoryl end product can be linked with the signal indicator by Ti(4+). The redox ferrocene group on the SWCNTs is grafted to the electrode and generates the electrochemical signal, the intensity of which is proportional to the activity of T4 PNK. This assay can measure activity of T4 PNK down to 0.01 UmL(-1). The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.     

Quencher-free hairpin probes for real-time detection of T4 polynucleotide kinase activity

Traditional methods of assaying polynucleotide kinase (PNK) activity are discontinuous, time-consuming, and laborious. Here we report a new quencher-free approach to real-time monitoring of PNK activity using a 2-aminopurine probe. When the 2-aminopurine probe was 5′-phosphorylated by PNK, it could be efficiently degraded by lambda exonuclease to release free 2-aminopurine molecules and generate a fluorescence signal. This method not only provides a universal approach to real-time monitoring of PNK activity, but also shows great potential for screening suitable inhibitor drugs for PNK.     

Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5'-attached to oligonucleotides.

The ability of T4 polynucleotide kinase (PNK) to phosphorylate non-nucleosidic moieties 5'-attached to oligodeoxynucleotides (ODNs) has been investigated. Non-nucleosidic phosphoramidite units were prepared from ethane-1,2-diol and propane-1,3-diol backbones. Some of them corresponded to pure enantiomers. They were used to obtain the corresponding 5'-end modified oligothymidylates X(pdT)10. The free primary hydroxyl of the non-nucleosidic moieties (X) of these oligomers was phosphorylated by PNK. We report the stereoselective phosphorylation of the L form of the 5'-end attached non-nucleosidic chiral fragments; the non-chiral moieties were completely phosphorylated. Dimers of glycerol analogue and thymidine 3'-phosphate were not recognized by PNK and the shortest modified ODN able to be phosphorylated was a trinucleotide X(pdT)3. A modified X(pdT)10, bearing a cyclic abasic site (X) at its 5'-end, was prepared by chemical synthesis from 1,2-dideoxyribose phosphoramidite and was phosphorylated with a 90% yield.     

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme

T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 A crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5' OH acceptor side that can accommodate a single-stranded polynucleotide. The active site is composed of essential side chains that coordinate the beta phosphate of the NTP donor and the 3' phosphate of the 5' OH acceptor, plus a putative general acid that activates the 5' OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.     

The stereochemical course of thiophosphoryl group transfer catalyzed by T4 polynucleotide kinase

The stereochemical course of the phosphoryl transfer reaction catalyzed by T4 polynucleotide kinase has been determined using the chiral ATP analog, (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate). T4 polynucleotide kinase catalyzes the transfer of the gamma-thiophosphoryl group of (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate) to the 5'-hydroxyl group of ApA to give the thiophosphorylated dinucleotide adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine. A sample of adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine was subjected to venom phosphodiesterase digestion. The resulting adenosine-5'-[18O]phosphorothioate was shown to have the Rp configuration, thus indicating that the thiophosphoryl transfer reaction occurs with overall inversion of configuration of phosphorus.     

Partial characterization of an endonuclease activity which appears in nuclease free T4 polynucleotide kinase.

A nuclease activity has been found to appear in preparations of T4 induced polynucleotide kinase which had originally been nuclease free. The nuclease introduced random nicks into T7 DNA suggesting that it was an endonuclease. Destabilization of the kinase molecule by osmotic shock or by the removal of reducing agents, ATP or salts was shown to stimulate the endonuclease appearance. The molecular weight was found to be 32,000 +/- 10% by gel filtration on G100 Sephadex. The nuclease was active over a wide pH range from pH 5.0 to pH 9.2 in a number of buffer systems and required MgCl2 and reducing agent for maximum activity. Sodium azide did not affect the nuclease appearance.     

A rapid purification of bovine testicular hyaluronidase by chromatography on dermatan sulphate-substituted 1,6-diaminohexane--sepharose 4B.

Dimensionless apparent ionization constants of charged low-molecular-weight species may be obtained from paper-electrophoretic data at 20-25 degrees C with buffers (I0.1-0.5) of measured pH (1.5-12.5) containing oxalate ions. Relative mobilities rather than absolute mobilities were measured by using glycerol and m-nitrobenzenesulphonate respectively as standards of zero and unit mobility. Application of the procedure to ionizations of adenine, adenosine, 2'-deoxyadenosine, 3'-deoxyadenosine, 3':5'-cyclic AMP, ADP, ADP-glucose-agrocin 84 and ATP is described.