Structure of a tRNA repair enzyme and molecular biology workhorse: T4 polynucleotide kinase

T4 phage polynucleotide kinase (PNK) was identified over 35 years ago and has become a staple reagent for molecular biologists. The enzyme displays 5'-hydroxyl kinase, 3'-phosphatase, and 2',3'-cyclic phosphodiesterase activities against a wide range of substrates. These activities modify the ends of nicked tRNA generated by a bacterial response to infection and facilitate repair by T4 RNA ligase. DNA repair enzymes that share conserved motifs with PNK have been identified in eukaryotes. PNK contains two functionally distinct structural domains and forms a homotetramer. The C-terminal phosphatase domain is homologous to the L-2-haloacid dehalogenase family and the N-terminal kinase domain is homologous to adenylate kinase. The active sites have been characterized through structural homology analyses and visualization of bound substrate.     

The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase

Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.     

The structural basis for substrate recognition by mammalian polynucleotide kinase 3' phosphatase

Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.     

Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3'-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage.

Mammalian polynucleotide kinases catalyze the 5'-phosphorylation of nucleic acids and can have associated 3'-phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage. The sequences of three tryptic peptides from a bovine 60-kDa polypeptide that correlated with 5'-DNA kinase and 3'-phosphatase activities identified human and murine dbEST clones. The 57.1-kDa conceptual translation product of this gene, polynucleotide kinase 3'-phosphatase (PNKP), contained a putative ATP binding site and a potential 3'-phosphatase domain with similarity to L-2-haloacid dehalogenases. BLAST searches identified possible homologs in Caenorhabditis elegans, Schizosaccharomyces pombe, and Drosophila melanogaster. The gene was localized to chromosome 19q13.3-13.4. Northern analysis indicated a 2-kilobase mRNA in eight human tissues. A glutathione S-transferase-PNKP fusion protein displayed 5'-DNA kinase and 3'-phosphatase activities. PNKP is the first gene for a DNA-specific kinase from any organism. PNKP expression partially rescued the sensitivity to oxidative damaging agents of the Escherichia coli DNA repair-deficient xth nfo double mutant. PNKP gene function restored termini suitable for DNA polymerase, consistent with in vivo removal of 3'-phosphate groups, facilitating DNA repair.     

The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks

The dual function mammalian DNA repair enzyme, polynucleotide kinase (PNK), facilitates strand break repair through catalysis of 5′-hydroxyl phosphorylation and 3′-phosphate dephosphorylation. We have examined the relative activities of the kinase and phosphatase functions of PNK using a novel assay, which allows the simultaneous characterization of both activities in processing nicks and gaps containing both 3′-phosphate and 5′-hydroxyl. Under multiple turnover conditions the phosphatase activity of the purified enzyme is significantly more active than its kinase activity. Consistent with this result, phosphorylation of the 5′-hydroxyl is rate limiting in cell extract mediated-repair of a nicked substrate. On characterizing the effects of individually mutating the two active sites of PNK we find that while site-directed mutagenesis of the kinase domain of PNK does not affect its phosphatase activity, disruption of the phosphatase domain also abrogates kinase function. This loss of kinase function requires the presence of a 3′-phosphate, but it need not be present in the same strand break as the 5′-hydroxyl. PNK preferentially binds 3′-phosphorylated substrates and DNA binding to the phosphatase domain blocks further DNA binding by the kinase domain.     

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.     

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.     

Further purification and characterization of the DNA 3'-phosphatase from rat-liver chromatin which is also a polynucleotide 5'-hydroxyl kinase

The DNA 3'-phosphatase activity of rat-liver chromatin has been purified. A DNA 5'-hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5'-hydroxyl kinase and a 3'-phosphatase. An additional argument is that ATP, which does not influence the rate of the 3'-phosphatase reaction but which is a cosubstrate of the 5'-hydroxyl kinase, protects the 3'-phosphatase activity against thermal denaturation and trypsin digestion. The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion. The rates of 3'-dephosphorylation and 5'-phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3'-phosphatase has no action on 3'-monodeoxynucleotide, but it hydrolyzes the 3'-phosphate in dinucleotides. The Km of the 3'-phosphatase is 0.548 microM. The Km (5'-OH) and Km (ATP) of the 5'-hydroxyl kinase are about 3.9 microM and 0.69 microM respectively. The chromatin enzyme is unable to hydrolyze 3'-phosphoglycolate ends in DNA.     

Highly sensitive fluorescence assay of T4 polynucleotide kinase activity and inhibition via enzyme-assisted signal amplification

DNA phosphorylation catalyzed by polynucleotide kinase (PNK) is an indispensable process in the repair, replication, and recombination of nucleic acids. Here, an enzyme-assisted amplification strategy was developed for the ultrasensitive monitoring activity and inhibition of T4 PNK. A hairpin oligonucleotide (hpDNA) was designed as a probe whose stem can be degraded from the 5′ to 3′ direction by lambda exonuclease (λ exo) when its 5′ end is phosphorylated by PNK. So, the 3′ stem and loop part of hpDNA was released as an initiator strand to open a molecular beacon (MB) that was designed as a fluorescence reporter, leading to a fluorescence restoration. Then, the initiator strand was released again by the nicking endonuclease (Nt.BbvCI) to hybridize with another MB, resulting in a cyclic reaction and accumulation of fluorescence signal. Based on enzyme-assisted amplification, PNK activity can be sensitively and rapidly detected with a detection limit of 1.0 × 10⁻⁴ U/ml, which is superior to those of most existing approaches. Furthermore, the application of the proposed strategy for screening PNK inhibitors also demonstrated satisfactory results. Therefore, it provided a promising platform for monitoring activity and inhibition of PNK as well as for studying the activity of other nucleases.     

Highly specific fluorescence detection of T4 polynucleotide kinase activity via photo-induced electron transfer

Sensitive and reliable study of the activity of polynucleotide kinase (PNK) and its potential inhibitors is of great importance for biochemical interaction related to DNA phosphorylation as well as development of kinase-targeted drug discovery. To achieve facile and reliable detection of PNK activity, we report here a novel fluorescence method for PNK assay based on a combination of exonuclease cleavage reaction and photo-induced electron transfer (PIET) by using T4 PNK as a model target. The fluorescence of 3′-carboxyfluorescein-labeled DNA probe (FDNA) is effectively quenched by deoxyguanosines at the 5′ end of its complementary DNA (cDNA) due to an effective PIET between deoxyguanosines and fluorophore. Whereas FDNA/cDNA hybrid is phosphorylated by PNK and then immediately cleaved by lambda exonuclease (λ exo), fluorescence is greatly restored due to the break of PIET. This homogeneous PNK activity assay does not require a complex design by taking advantage of the quenching ability of deoxyguanosines, making the proposed strategy facile and cost-effective. The activity of PNK can be sensitively detected in the range of 0.005 to 10 U mL⁻¹ with a detection limit of 2.1 × 10⁻³ U mL⁻¹. Research on inhibition efficiency of different inhibitors demonstrated that it can be explored to evaluate inhibition capacity of inhibitors. The application for detection of PNK activity in complex matrix achieved satisfactory results. Therefore, this PIET strategy opens a promising avenue for studying T4 PNK activity as well as evaluating PNK inhibitors, which is of great importance for discovering kinase-targeted drugs.     

Characterization of polynucleotide kinase/phosphatase enzymes from Mycobacteriophages omega and Cjw1 and vibriophage KVP40

Coliphage T4 Pnkp is a bifunctional polynucleotide 5'-kinase/3'-phosphatase that catalyzes the end-healing steps of a RNA repair pathway. Here we show that mycobacteriophages Omega and Cjw1 and vibriophage KVP40 also encode bifunctional Pnkp enzymes consisting of a proximal 5'-kinase module with an essential P-loop motif, GXGK(S/T), and a distal 3'-phosphatase module with an essential acyl-phosphatase motif, DX- DGT. Biochemical characterization of the viral Pnkp proteins reveals several shared features, including an alkaline pH optimum for the kinase component, an intrinsic RNA kinase activity, and a homotetrameric or homodimeric quaternary structure, that distinguish them from the monomeric DNA-specific phosphatase/kinase enzymes found in mammals and fission yeast. Whereas the phage 5'-kinases differ from each other in their preferences for phosphorylation of 5' overhangs, blunt ends, or recessed ends, none of them displays the preference for recessed ends reported for mammalian DNA kinase. We hypothesize that Pnkp provides phages that have it with a means to evade an RNA-damaging antiviral host response. Genetic complementation of the essential end-healing steps of yeast tRNA splicing by the Omega and Cjw1 Pnkp enzymes establishes their capacity to perform RNA repair reactions in vivo. A supportive correlation is that Omega and Cjw1, which are distinguished from other mycobacteriophages by their possession of a Pnkp enzyme, are also unique among the mycobacteriophages in their specification of putative RNA ligases.     

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.     

Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining

The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.     

Simultaneous detection of kinase and phosphatase activities of polynucleotide kinase using molecular beacon probes

Phosphorylation and dephosphorylation of DNA by polynucleotide kinase (PNK) has an important role in DNA damage repair, replication, and recombination. Traditionally, it is assayed by denaturing gel electrophoresis and autoradiography, which are tedious and not sensitive. We report on the development of a sensitive and simple method for PNK assay based on DNA ligation using a molecular beacon. Enzyme activity of PNK is measured down to a limit of 0.002 unit/ml. The method not only provides a universal platform for simultaneous monitoring of kinase and phosphatase activities, but also shows great potential in biological research, drug discovery, and clinical diagnostics.     

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.     

Characterization of a baculovirus enzyme with RNA ligase, polynucleotide 5'-kinase, and polynucleotide 3'-phosphatase activities

The end-healing and end-sealing steps of the phage T4-induced RNA restriction-repair pathway are performed by two separate enzymes, a bifunctional polynucleotide 5'-kinase/3'-phosphatase and an ATP-dependent RNA ligase. Here we show that a single trifunctional baculovirus enzyme, RNA ligase 1 (Rnl1), catalyzes the identical set of RNA repair reactions. Three enzymatic activities of baculovirus Rnl1 are organized in a modular fashion within a 694-amino acid polypeptide consisting of an autonomous N-terminal RNA-specific ligase domain, Rnl1-(1-385), and a C-terminal kinase-phosphatase domain, Rnl1-(394-694). The ligase domain is itself composed of two functional units. The N-terminal module Rnl1-(1-270) contains essential nucleotidyltransferase motifs I, IV, and V and suffices for both enzyme adenylylation (step 1 of the ligation pathway) and phosphodiester bond formation at a preactivated RNA-adenylate end (step 3). The downstream module extending to residue 385 is required for ligation of a phosphorylated RNA substrate, suggesting that it is involved specifically in the second step of the end-joining pathway, the transfer of AMP from the ligase to the 5'-PO(4) end to form RNA-adenylate. The end-healing domain Rnl1-(394-694) consists of a proximal 5'-kinase module with an essential P-loop motif ((404)GSGKS(408)) and a distal 3'-phosphatase module with an essential acylphosphatase motif ((560)DLDGT(564)). Our findings have implications for the evolution of RNA repair systems and their potential roles in virus-host dynamics.     

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.     

T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product.

The T4 gene (pseT) for polynucleotide kinase (pnk) has been cloned in lambda. Induction of a lambda E-W-S-cI857 prophage in which the pseT gene can be transcribed from the late lambda promoter, PR1, leads to greater than 100-fold amplification of pnk activity; pnk comprises approximately 7% of the total soluble cell protein. The purified enzyme, as expected, is both a 5'-kinase and a 3'-phosphatase. The amino acid sequence deduced from an open reading frame identified as the pseT gene contains a sequence which corresponds particularly well with that part of the adenine nucleotide binding site of adenylate kinase shown to form a flexible loop. A deletion mutant that lacks 5'-kinase activity, and possibly also 3'-phosphatase activity, has lost two amino acids from within the proposed loop structure. A second region of the pnk sequence shares homology with phosphoglycerate kinase, yeast inorganic pyrophosphatase and histone 2b from various organisms.     

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.