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Originally published In Press as doi:10.1074/jbc.M603403200 on July 6, 2006

J. Biol. Chem., Vol. 281, Issue 38, 28023-28032, September 22, 2006
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Identification of a Novel Binding Motif in Pyrococcus furiosus DNA Ligase for the Functional Interaction with Proliferating Cell Nuclear Antigen*Formula

Shinichi Kiyonari{ddagger}, Kohei Takayama{ddagger}, Hirokazu Nishida§, and Yoshizumi Ishino{ddagger}1

From the {ddagger}Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, and BIRD-Japan Science and Technology Agency, 6-10-1 Hakozaki, Fukuoka-shi, Fukuoka 812-8581, Japan and §Central Research Laboratories, Hitachi Limited, 1-280 Higashi-koigakubo, Kokubunji, Tokyo 185-8581, Japan

Received for publication, April 10, 2006 , and in revised form, May 22, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA ligase is an essential enzyme for all organisms and catalyzes a nick-joining reaction in the final step of the DNA replication, repair, and recombination processes. Herein, we show the physical and functional interaction between DNA ligase and proliferating cell nuclear antigen (PCNA) from the hyperthermophilic Euryarchaea Pyrococcus furiosus. The stimulatory effect of P. furiosus PCNA on the enzyme activity of P. furiosus DNA ligase was observed not at low ionic strength, but at a high salt concentration, at which a DNA ligase alone cannot bind to a nicked DNA substrate. On the basis of mutational analyses, we identified the amino acid residues that are critical for PCNA binding in a loop structure located in the N-terminal DNA-binding domain of P. furiosus DNA ligase. We propose that the pentapeptide motif QKSFF is involved in the PCNA-interacting motifs, in which Gln and the first Phe are especially important for stable binding with PCNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA ligases catalyze nick-sealing reactions via three nucleotidyl transfer steps as described in recent review articles (1–4). In the first step, DNA ligases form a covalent enzyme-AMP intermediate by reacting with ATP or NAD+ as a cofactor (Step 1). In the second step, DNA ligases recognize the substrate DNA, and the AMP is subsequently transferred from the ligases to the 5'-phosphate terminus of the DNA to form a DNA-adenylate intermediate (Step 2). Then, in the final step, the 5'-DNA-adenylate intermediate is attacked by the adjacent 3'-hydroxy group of the DNA to form a phosphodiester bond (Step 3). Three genes (LIG1, LIG3, and LIG4) encoding ATP-dependent DNA ligases have been identified in the human genome to date. Human DNA ligase I (Lig I)2 is a replicative enzyme that joins Okazaki fragments during the DNA replication process. It is well known that many eukaryotic proteins involved in DNA replication, DNA repair, and cell cycle control interact with a DNA sliding clamp, proliferating cell nuclear antigen (PCNA) (reviewed in Refs. 5–7). Extensive studies of the PCNA-interacting proteins revealed the existence of a consensus sequence called the PCNA-interacting protein (PIP) box (5). The PIP box consists of the sequence QXXhXXaa, where X represents any amino acid, h represents hydrophobic residues (e.g. Leu, Ile, or Met), and a represents aromatic residues (e.g. Phe, Tyr, or Trp). Furthermore, it has been proposed that other sites of the interacting protein can participate in PCNA binding. For example, a conserved pair of Lys and Ala residues was identified as a PCNA-binding motif (KA box) using a random peptide display library (8). However, the importance of the KA box is not obvious because a detailed biochemical analysis of the motif has not been performed to date. In Escherichia coli, the corresponding DNA sliding clamp is the beta-subunit of the DNA polymerase III holoenzyme (henceforth referred to as the beta-clamp), which forms a toroidal dimeric structure (9). A bioinformatics approach revealed that a pentapeptide motif (consensus QL(S/D)LF) plays an important role in binding to the beta-clamp (10).

In higher eukaryotes, human Lig I reportedly forms a stable complex with a PCNA trimer that is topologically linked to duplex DNA via an N-terminal PIP box motif (11, 12). The structures of eukaryotic DNA ligases can be divided into two major domains, an N-terminal non-catalytic domain and a C-terminal catalytic domain, which consists of an adenylation domain and an OB (oligonucleotide/oligosaccharide binding)-fold domain (3, 13). The crystal structure of human Lig I in complex with a nicked DNA and biochemical analyses of the enzyme revealed that the non-catalytic domain provides most of the DNA binding affinity (14), and therefore, this domain is called the N-terminal DNA-binding domain (DBD). Although several groups have characterized the physical interactions between human Lig I and PCNA, no stimulatory or inhibitory effect on nick-joining activities has been observed in vitro (11, 15). In contrast, a stimulatory effect of PCNA was also reported (16). Thus, the detailed interaction mode between human PCNA and Lig I is somewhat unclear.

In Archaea, the third domain of life, a single homolog of eukaryotic Lig I has been identified (17–21). Interestingly, although most of the archaeal replicative enzymes have a eukaryotic PIP box at their C termini, no clear PCNA-binding motif has been observed in the sequences of archaeal DNA ligases (5, 22). Recently, a physical and functional interaction between PCNA and DNA ligase from Sulfolobus solfataricus was reported (23). In the S. solfataricus DNA ligase, PIP box-like motifs were proposed to exist in the N-terminal region. Furthermore, a mutant S. solfataricus DNA ligase lacking 30 amino acids at the N terminus cannot interact with S. solfataricus PCNA (23, 24). However, it has not been determined whether the proposed motifs are actually important for the interaction with PCNA.

Here, we show a physical and functional interaction between PCNA and DNA ligase from the hyperthermophilic Euryarchaea Pyrococcus furiosus. The stimulatory effect of P. furiosus PCNA (PfuPCNA) on the nick-joining reaction of P. furiosus DNA ligase (PfuLig) was observed under physiological conditions at an extremely high salt concentration (0.5–0.6 M). Furthermore, we show that the pentapeptide sequence QKSFF in the DBD of PfuLig plays an important role in binding to PfuPCNA. Interestingly, this motif is located in a loop connecting two {alpha}-helices in the DBD, but is not at the N or C terminus of PfuLig, based on our crystal structure.3 We propose a novel PCNA-binding motif, which may be located inside, but not at the termini, of the PCNA-interacting proteins.


Figure 1
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  		FIGURE 1.
ATP dependence and domain structure of PfuLig. A, adenylyltransferase assay of wild-type (WT) and K249A mutant PfuLig proteins. Purified enzymes (64 kDa) were mixed with [{alpha}-32P]ATP in the ligation reaction buffer as described under "Experimental Procedures." The reaction mixtures were fractionated by SDS-PAGE, and the adenylylated band was detected by autoradiography. T4 DNA ligase (62 kDa) was used as a positive control of a well known ATP-dependent DNA ligase. The positions and sizes (in kilodaltons) of maker polypeptides are indicated on the left. B, cofactor dependence of the PfuLig ligation activity. Deadenylylated PfuLig (10 fmol) was subjected to the ligation reaction with 500 fmol of nicked DNA as described under "Experimental Procedures." The ligation efficiencies were compared with those of different cofactors: ATP, ADP, AMP, and NAD+. C, domain structure of PfuLig. The domain structure of PfuLig is schematically drawn. AdD, adenylylation domain. Two PfuLig truncation mutants were constructed based on the domain structure. D, comparison of the ligation abilities of the wild-type (10 fmol), K249A (10 fmol), DBD (1 pmol), and CD (adenylylation domain + OB-fold domain; 1 pmol) proteins. The ligation reactions (containing 100 fmol of nicked DNA) were analyzed by denaturing PAGE, followed by autoradiography. The upper band on the 22-mer deoxynucleotide substrate is from the adenylylated form (AppDNA), which is the intermediate observed at the second step. nt, nucleotides.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning the PfuLig Gene and Its Mutants—The DNA ligase gene (lig) was amplified by PCR directly from P. furiosus genomic DNA using oligonucleotides 5'-GGCCATGGGTTATCTGGAGCTTGCTCAAC-3' and 5'-GCGGATCCTTAGCTTTCCACTTTTCCTTTCATC-3' as the forward and reverse primers, respectively. The amplified gene was cloned into the pGEM-T Easy vector (Promega), and its nucleotide sequence was confirmed. The cloned gene was digested by NcoI-BamHI and inserted into the corresponding sites of pET-21d (Novagen). The resultant plasmid was designated pET-Lig. Amino acid substitutions and N-terminal truncations were introduced into the lig gene on the pET-Lig plasmid by PCR using the appropriate primers.4

Overproduction and Purification of PfuLig Proteins—To obtain recombinant PfuLig, E. coli BL21-CodonPlusTM(DE3)-RIL cells (Stratagene) carrying pET-Lig were grown in 1 liter of LB medium containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol at 37 °C. The cells were cultured to A600 = 0.40, and expression of the lig gene was induced by adding isopropyl beta-D-thiogalactopyranoside to a final concentration of 1 mM and continuing the culture for 5 h at 37 °C. After cultivation, the cells were harvested and disrupted by sonication in buffer A (50 mM Tris-HCl (pH 8.0), 0.5 mM dithiothreitol (DTT), 0.1 mM EDTA, and 10% glycerol). The soluble cell extract obtained by centrifugation at 12,000 x g for 20 min was heated at 80 °C for 20 min. The heat-resistant fraction obtained by centrifugation was treated with 0.15% polyethyleneimine to remove the nucleic acids. The soluble proteins were precipitated by 80% saturated ammonium sulfate. The precipitate was resuspended in buffer B (50 mM Tris-HCl (pH 8.0), 1 M (NH4)2SO4, 0.5 mM DTT, 0.1 mM EDTA, and 10% glycerol) and subjected to chromatography on a HiTrap phenyl column (Amersham Biosciences). The proteins were eluted at 0 M ammonium sulfate, and the eluted proteins were dialyzed against buffer A. The dialysate was loaded onto a HiTrap heparin column (Amersham Biosciences), and the proteins were eluted at 0.3–0.35 M sodium chloride. The eluted proteins were dialyzed against buffer A, and the dialysate was subjected to chromatography on a HiTrap Q column (Amersham Biosciences). The proteins were eluted at 0.1–0.15 M sodium chloride, pooled, and stored at 4 °C. The mutant PfuLig proteins prepared in this study were purified by the same procedures. The purity of each protein used in this study was evaluated by SDS-PAGE. No extra band was detected by Coomassie Brilliant Blue staining of the gel containing 2 µg of each purified protein. The protein concentrations were calculated by measuring the absorbance at 280 nm. The theoretical molecular absorption coefficient of the molecule was calculated based on its tryptophan and tyrosine content.

Preparation of the Deadenylylated Enzyme—Purified wild-type PfuLig protein (1.5 nmol) was incubated with an excess amount of pyrophosphoric acid (10 mM) in 150 µl of reaction buffer (20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Tween 20) at 37 °C for 1 h. The deadenylylated enzyme was purified by applying the reaction mixture to a MicroSpin G-25 column (Amersham Biosciences) pre-equilibrated with buffer A.

Adenylyltransferase Assay—Purified wild-type or K249A mutant PfuLig protein (15 pmol) was incubated with 0.05 µCi of [{alpha}-32P]ATP in 20 µl of reaction buffer at 55 °C for 20 min. T4 DNA ligase (9 units; Promega) was incubated with 0.05 µCi of [{alpha}-32P]ATP in a reaction mixture (20 µl) containing 30 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT, and 5% polyethylene glycol (Mr cutoff of 8000) at 35 °C for 20 min. The products were analyzed by 10% SDS-PAGE. After electrophoresis, the gels were dried and scanned using a Fujifilm FLA5000 image analyzer to detect the Ado-[32P]DNA ligase adducts.

DNA Ligation Assay—The substrate DNA used in the ligation assay was a 49-bp DNA duplex containing a single nick at the center. The 22-mer deoxynucleotide (5'-AATTCGTGCAGGCATGGTAGCT-3'), which was labeled with 32Patthe 5' terminus, and the 27-mer deoxynucleotide (5'-AGCTATGACCATGATTACGAATTGCTT-3') were annealed to the 49-mer deoxyoligonucleotide with a complementary sequence in 40 mM Tris acetate (pH 7.8) and 0.5 mM magnesium acetate. The purified PfuLig proteins (at different concentrations for each experiment, as described in the figure legends) were incubated with the nicked DNA substrate (5 nM), prepared as described above, in 20 µl of ligation buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 0.01 mM ATP, 0.1% Tween 20, and 0.1 mg/ml bovine serum albumin at 60 °C for 15 min. Reactions were initiated by the addition of enzyme and terminated with 5 µl of stop solution containing 98% formamide, 10 mM EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol. Samples were heated at 100 °C for 5 min and chilled rapidly on ice prior to loading onto a 10% polyacrylamide gel containing 8 M urea. After electrophoresis, the gels were dried and scanned using the FLA5000 image analyzer to detect the 32P-labeled DNA. Three independent experiments were carried out in succession for each ligation condition required in this study, and the S.E. values are shown as vertical lines on the plots in each graph.


Figure 2
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  		FIGURE 2.
Physical interaction between PfuLig and PfuPCNA. SPR analysis was performed using a Biacore system to detect a physical interaction between PfuLig and PfuPCNA. Purified PfuLig was immobilized on a Biacore sensor chip, and purified PfuPCNA (3 µM) was loaded. Wild-type (WT) PfuPCNA and D143A/D147A mutant PfuPCNA, which cannot form a stable trimeric ring structure, were used to investigate their affinities for PfuLig. The equilibrium constant (KD) was calculated from the obtained sensorgram.

 
Surface Plasmon Resonance Analysis—A Biacore system was used to study the physical interaction between PfuLig and PfuPCNA. Highly purified recombinant PfuLig or PfuPCNA (25) was fixed on a CM5 sensor chip (research grade; Biacore) according to the manufacturer's recommendations. To measure the kinetic parameters, various concentrations of PfuPCNA were applied to immobilized PfuLig. All measurements were performed at a continuous flow rate of 30 µl/min in buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween 20. At the end of each cycle, the bound protein was removed by washing with 2 M NaCl. The kinetic constants for PfuPCNA binding to PfuLig were determined from the association and dissociation curves of the sensorgrams using the BIAevaluation program (Biacore).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Biochemical Properties of PfuLig—PfuLig has already been characterized and is commercially available, mainly as a reagent for ligase chain reactions (Stratagene) (38). In this study, we cloned and purified PfuLig independently and constructed mutant proteins to analyze the structure-function relationships of this enzyme. It was predicted from the primary amino acid sequence similarity that PfuLig is an ATP-dependent DNA ligase. Therefore, we constructed the mutant lig gene encoding K249A mutant PfuLig, in which the lysine at the predicted adenylylation site was substituted with alanine, in parallel with the gene for wild-type PfuLig and tested their adenylyltransferase activities in the presence of ATP. As shown in Fig. 1A, wild-type PfuLig could form a covalent enzyme-AMP intermediate by reacting with [{alpha}-32P]ATP as a cofactor, but no adenylyltransferase activity was observed with the K249A mutant protein. It was reported that some thermophilic DNA ligases from Archaea utilize ADP (26) or NAD+ (19, 27) as a cofactor. However, we detected a distinct activity of PfuLig in the presence of ATP, but not ADP, AMP, and NAD+ (Fig. 1B). A very small amount of ligation product was detected by the reaction with ADP. This result is the same as in the case of DNA ligase from Pyrococcus horikoshii in a recent report (21). We think that the ligation reaction may be derived from contaminant ATP (1.16%) in our ADP reagent (Oriental Yeast Co., Ltd., Osaka, Japan) according to the manufacturer's certificate, and therefore, we concluded that ADP is not an appropriate cofactor for PfuLig, as Keppetipola and Shuman (21) described for P. horikoshii DNA ligase.


Figure 3
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  		FIGURE 3.
Salt sensitivity of PfuLig and effect of PfuPCNA on ligation ability. A, comparison of ligation efficiencies at various KCl and potassium glutamate (K-Glu) concentrations in the reaction mixture as described under "Experimental Procedures" with 20 fmol of PfuLig and 100 fmol of nicked DNA in the absence of PfuPCNA. B and C, salt dependence of PfuPCNA-mediated stimulation of PfuLig activity. The ligation reactions of PfuLig (2 fmol) and the nicked DNA (100 fmol) were done with increasing concentrations of KCl (B) and potassium glutamate (C) in the presence or absence of PfuPCNA. The concentration of PfuPCNA was calculated as the trimeric form.

 
Our crystallographic study of wild-type PfuLig revealed that the protein consists of three distinct domains, the N-terminal DBD, the middle adenylylation domain, and the C-terminal OB-fold domain.3 The last two domains are commonly called the core catalytic domain (CD), which is conserved in one branch of the nucleotidyltransferase superfamily, containing DNA ligase, RNA ligase, and mRNA capping enzymes (reviewed in Ref. 4). We made truncated PfuLig proteins, including the DBD from amino acids 1 to 218 (N-terminal domain) and the CD from amino acids 219 to 561 (middle and C-terminal domains) to investigate the functions of each domain (Fig. 1C). A nick joining assay was performed using the wild-type enzyme and the mutant PfuLig proteins (K249A, DBD, and CD) (Fig. 1D). The CD protein could not complete the nick-sealing reaction, even at high enzyme concentrations. A very small amount of ligation product was observed when the CD protein was added at a concentration 100-fold higher than that of the wild-type protein (Fig. 1D, CD lane). The accumulation of DNA-adenylate intermediate products implied that the CD protein exhibits a lower activity at the "Step 3" reaction. The CD protein shares a structural similarity with the full-length Chlorella virus DNA ligase, which is the smallest ATP-dependent DNA ligase with a distinct activity in vitro (28), and therefore, it is very interesting to investigate why the CD from PfuLig lacks most of the enzyme activity. This result indicates the importance of the DBD for the overall ligation activity of PfuLig, and it can be predicted from the structural similarity that the contribution of the DBD to the ligation reaction is also conserved in the eukaryotic DNA ligases.


Figure 4
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  		FIGURE 4.
The N-terminal DBD of PfuLig is essential for PfuPCNA binding. Physical interactions with PfuPCNA were compared among the wild-type (WT) and two truncated (DBD and CD) PfuLig proteins using the Biacore system. Purified PfuPCNA was immobilized on a Biacore sensor chip, and purified PfuLig proteins (3 µM) were loaded in reverse to the experiment shown in Fig. 2.

 
PfuLig Can Interact with Both Monomeric and Trimeric PCNA Proteins—To determine the physical interaction between PfuLig and PfuPCNA, we first used an immunoprecipitation method. The PfuPCNA and PfuLig proteins were incubated together and then precipitated by each antiserum. However, significant interactions between them were not detected under several experimental conditions, probably because the protein-protein interaction was too weak to be detected by an immunoprecipitation method (data not shown). We therefore performed surface plasmon resonance (SPR) experiments to analyze the weak DNA ligase-PCNA interactions. Full-length PfuLig was immobilized on the Biacore CM5 sensor chip, and subsequently, wild-type PfuPCNA and D143A/D147A mutant PfuPCNA, which is unable to form a stable toroidal structure in solution and thus cannot stimulate P. furiosus DNA polymerase B activity (29), were injected at different concentrations. The physical interactions between immobilized PfuLig and the two PCNA proteins were identified by the SPR sensorgram (Fig. 2). The calculated equilibrium constants (KD) for wild-type PCNA and the D143A/D147A mutant were 1.1 x 10–7 and 1.4 x 10–6 M, respectively. The KD values reported here are comparable with that for the human PCNA-p21 interaction determined by SPR analysis (KD = 3.2 x 10–7 M) (30). These findings suggest that the toroidal structure is not necessarily required to form a stable PfuLig·PfuPCNA complex in vitro.


Figure 5
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  		FIGURE 5.
Search for the candidate amino acid sequence motifs in the DNA ligase proteins. A, based on the proposed binding region (black boxes) in S. solfataricus DNA ligase (SsoLig) and the secondary structure of PfuLig, two PfuLig truncation mutants were prepared to investigate the interaction site of PfuLig and PfuPCNA. Asterisks and colons indicate identity and similarity, respectively, of the aligned residues. B, the amino acid sequence of the DBD was examined carefully, and a new PIP box-like motif was found, in addition to the candidate KA box, as shown by the black box. The regions containing these candidate motifs were aligned with the human Lig I (huLig I) sequence. This PIP box-like motif is located in the loop structure connecting the sixth and seventh{alpha}-helices (residues 103–107) of the PfuLig crystal structure. In the crystal structure of human Lig I, part of the corresponding region is disordered (residues 385–392, indicated by the dashed line) (14). C, the ligation activities of wild-type (WT) PfuLig, PfuLig{Delta}N14, PfuLig{Delta}N32, and F106A/F107A mutant PfuLig were compared. Various amount of PfuLig proteins as indicated were incubated with 100 fmol of nicked DNA in the reaction mixture as described under "Experimental Procedures." The ligation efficiency is plotted as a function of the enzyme concentration (logarithmically shown on the horizontal axis). D, shown is the stimulatory effect of PfuPCNA on the ligation ability of the mutant enzymes. Wild-type PfuLig or F106A/F107A mutant PfuLig (2 fmol), PfuLig{Delta}N14 (4 fmol), or PfuLig{Delta}N32 (60 fmol) was incubated with 100 fmol of nicked DNA and 0–150 nM PfuPCNA in a reaction mixture containing 0.2 M KCl as described under "Experimental Procedures." The ligation activity of PfuLig without KCl in the absence of PfuPCNA is plotted on the y axis as a control to show the basic PfuLig activity used for this experiment. E, the ligation abilities of the wild-type and K67A mutant PfuLig proteins were compared. The enzyme (2 fmol) was incubated with 100 fmol of nicked DNA and 0–150 nM PfuPCNA in a reaction mixture containing 0.2 M KCl as described under "Experimental Procedures," and the efficiencies are plotted as a function of PfuPCNA concentration. The plot on the y axis shows a control as explained for D.

 


Figure 6
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  		FIGURE 6.
The predicted motif in PfuLig is actually important for PfuPCNA binding. A, the position of the predicted PIP box-like motif in PfuLig (stick models surrounded by an oval) is shown in its overall crystal structure, flanked by a close-up stereo view in the box. B, using the PfuPCNA-immobilized Biacore sensor chip as shown in Fig. 4, the wild-type (WT) and Q103A, F106A, F107A, and F106A/F107A mutant (3 µM) PfuLig proteins were loaded to investigate their physical interactions with PfuPCNA. C, shown is the stimulatory effect of PfuPCNA on the ligation ability of mutant PfuLig proteins. PfuLig (2 fmol) was incubated with 100 fmol of nicked DNA and 0–150 nM PfuPCNA in a reaction mixture containing 0.2 M KCl as described under "Experimental Procedures." The ligation activity of PfuLig without KCl in the absence of PfuPCNA is plotted on the y axis as a control to show the basic PfuLig activity used for this experiment.

 
PfuPCNA Enhances the Ligation Activity of PfuLig at a Physiological Salt Concentration—It is well known that some hyperthermophilic Archaea contain strikingly high intracellular potassium ion concentrations. Based on the study of a Euryarchaea, Pyrococcus woesei, which was later proven to be a subspecies of P. furiosus (31), the potassium ion concentration in the hyperthermophilic archaeal cells was determined to range from 0.5 to 0.6 M (32). We initially examined the effect of increasing salt concentrations on the nick-joining activity of PfuLig by supplementing the reaction with KCl and potassium glutamate salts. A reduction in ligation activity was observed with each of these monovalent salts in a concentration-dependent manner, and ~90% inhibition was seen at a 200 mM salt concentration (Fig. 3A). A similar result was obtained in the enzyme assay using NaCl (data not shown). These observations are not specific for PfuLig, as the same phenomena were reported in characterizations of other DNA and RNA ligases (18, 27, 33).

To determine whether PfuPCNA can stimulate the ligation activity of PfuLig at the physiological ionic strength, the proteins were assayed at a broad range of salt concentrations. The stimulation effect of PfuPCNA on PfuLig was observed at 0.05–0.2 M KCl, but the effect was decreased at >0.2 M KCl (Fig. 3B). We performed the enzyme assay in the same manner with potassium glutamate salt, which has been reported to be an important factor contributing to the thermostability of archaeal proteins (34). As shown in Fig. 3C, the stimulation effect was observed at >0.2 M potassium glutamate and peaked at 0.3–0.4 M, and residual activity was detected near the physiological potassium concentrations (0.5–0.6 M). Before comparison of the effects of KCl and potassium glutamate concentrations on the PfuPCNA-dependent ligation activity of PfuLig, the ligation reactions were performed with increasing concentrations of PfuPCNA at constant salt concentrations, and the appropriate concentrations of PfuPCNA for each reaction in the presence of KCl or potassium glutamate were determined (supplemental Fig. S1). These results show that a chloride ion (Cl–) concentration of >0.2 M, but not this concentration of potassium ion (K+), had an inhibitory effect on the enzyme activity of PfuLig. The same result was observed in the characterization of a Holliday junction-resolving enzyme, Hjc, from P. furiosus, which exhibits maximum enzyme activity at 0.2 M KCl (35). For the P. furiosus enzymes that catalyze nucleic acid modification reactions, high Cl– concentrations may affect their activity.

A Novel PCNA-binding Site in the N-terminal DBD of PfuLig—To determine the region responsible for PCNA binding in PfuLig, we utilized the two truncation mutants (DBD and CD) as shown in Fig. 1C. The interactions between PfuPCNA and these truncated PfuLig proteins were examined qualitatively by SPR analysis. Wild-type PfuPCNA was immobilized on a Biacore CM5 sensor chip, and the two DNA ligase truncation mutants were then injected. Wild-type PfuLig and the DBD interacted with immobilized PfuPCNA to almost the same extent, but the CD had no binding ability (Fig. 4). This SPR analysis using immobilized PfuPCNA showed a very low number of resonance units compared with that shown in Fig. 2, in which PfuLig was immobilized. This phenomenon often happens in our experience of SPR analyses using PfuPCNA and its binding proteins. The difference probably depends on the direction of the proteins fixed on the sensor chip. Because of the relatively low number of resonance units (<250) observed in this experiment, the equilibrium constant (KD) was not determined. These findings suggest that the N-terminal DBD plays a critical role in the PCNA binding of PfuLig.

In Archaea, it was reported that S. solfataricus DNA ligase has a PCNA-binding site in its N-terminal region (23). To determine whether the same region of PfuLig is responsible for PCNA binding, we cloned and purified two N-terminally trun-cated mutants, PfuLig{Delta}N14 (amino acids 15–561) and PfuLig{Delta}N32 (amino acids 33–561), based on the crystal structure of PfuLig (Fig. 5A). In addition, we carefully examined the amino acid sequence of the DBD and found some regions that may be involved in interactions with PfuPCNA. Single amino acid substitution mutants in these regions were prepared to examine their effects on the PCNA interaction. These regions included a candidate KA box and a PIP box-like motif found in the DBD (Fig. 5B). Lys67 in the candidate KA box and the two aromatic residues in the PIP box-like sequence (Phe106 and Phe107) were examined by alanine substitutions. To test the stimulation activity of PCNA in the nick-sealing reaction (described above) under equivalent conditions, the relative activities of these mutant proteins were determined without PfuPCNA. The F106A/F107A mutant exhibited almost the same activity as wild-type PfuLig (Fig. 5C). The decreased activity observed in the two N-terminally truncated mutants (PfuLig{Delta}N14 and PfuLig{Delta}N32) revealed that the integrity of the DBD is important for the overall ligation activity. To determine the region responsible for PCNA binding, we tested the stimulation effect of PfuPCNA on these mutants. As a result, only the ligation activity of F106A/F107A was not stimulated by PfuPCNA (Fig. 5D). Furthermore, no effect of PfuPCNA was observed with increasing concentrations of F106A/F107A mutant PfuLig (data not shown). The PfuPCNA-dependent ligation ability of the K67A mutant was not different from that of the wild-type enzyme (Fig. 5E). We concluded that at least one of the two aromatic residues (Phe106 or Phe107) plays a crucial role in PCNA binding via a hydrophobic interaction.


Figure 7
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  		FIGURE 7.
Structural comparison of the novel PCNA-binding motif in the PfuLig crystal with the PIP box in the RFC large subunit in the cocrystal with PfuPCNA. The two motifs were extracted from each crystal structure. Cyan, PfuLig; orange, RFC large subunit (RFC-L). The side chains are depicted by stick models and are overlaid with the surface representations. Nitrogen (blue) and Oxygen (red) atoms were mapped onto the surface.

 
Gln103 and Phe106 of PfuLig Are Critical for the Functional Interaction with PfuPCNA—The PIP box-like sequence (103QKSFF107, described above) is located in a loop structure in the DBD based on the crystal structure of PfuLig (Fig. 6A). Using three single amino acid substitution mutants (Q103A, F106A, and F107A), we examined the detailed roles of each of these amino acid residues in the 103QKSFF107 sequence. The specific activities of these PfuLig mutants were confirmed to be the same (supplemental Fig. S2). The physical interactions between the mutant PfuLig proteins and immobilized PfuPCNA were analyzed by SPR analyses. The F106A mutant was not able to bind to PCNA, but the Q103A and F107A mutants showed very weak responses (50 resonance units) compared with wild-type PfuLig (240 resonance units) (Fig. 6B). Next, the stimulatory effect of PCNA on the ligation activity of these mutants was examined in vitro. As a result, the activities of the Q103A and F106A mutants were slightly stimulated by PfuPCNA, whereas the F107A mutant exhibited an intermediate response to PfuPCNA (Fig. 6C). These analyses indicated that Phe106 in the 103QKSFF107 sequence is the most important residue in the physical and functional interactions with PfuPCNA. Gln103 may stabilize the DNA ligase·PCNA complex after connecting PfuLig to PfuPCNA with Phe106. F107A mutant PfuLig, which possesses Gln103 and Phe106, showed very weak binding activity for PfuPCNA in the SPR analysis, comparable with that of Q103A mutant PfuLig, but a distinct response to PCNA was retained in the ligation assay. Further analyses will be required to understand the detailed role of Phe107 in the functional interaction between PfuLig and PfuPCNA.


Figure 8
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  		FIGURE 8.
The novel PCNA-binding motif is widely conserved in the archaeal and eukaryotic DNA ligases. A, alignment of the amino acid sequences of the putative loop regions containing the novel PIP motif in archaeal DNA ligases. To investigate the conservation of the PCNA-binding motifs identified in this study, a protein-protein BLAST search was carried out (www.ncbi.nlm.nih.gov/BLAST/) using the deduced DNA ligase sequences from the 26 completely sequenced archaeal genomes, involving 20 Euryarchaeota (black), 5 Crenarchaeota (red), and 1 Nanoarchaeota (blue) to date. The search results were manually evaluated, and the sequences were manually realigned. The basic residues, glutamine (Q), and phenylalanine (F) are highlighted in blue, magenta, and yellow, respectively. B, multiple alignment of the amino acid sequences of the loop regions in eukaryotic DNA ligases.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Functional Roles of the Conserved DBD in Eukaryotic DNA Ligases—As shown in our mutational analyses, the integrity of the DBD is important for the overall ligation activity of PfuLig itself (Fig. 1D). Furthermore, the other important function of the DBD is to interact with PCNA. The functional interaction between PfuLig and PfuPCNA seems to be stoichiometric. However, a large excess amount of PfuPCNA was required for stimulation of the ligation reaction by PfuLig (Fig. 5D). This inconsistency could be explained by the difficulty of PCNA loading onto the DNA fragment in the assay mixture. The PCNA trimer loads by diffusion onto the double-stranded DNA fragment over the ends without a clamp loader (replication factor C (RFC)) in this case. This is probably a limited process, and efficient loading requires a large stoichiometric excess of PCNA as discussed previously for human Lig I (16).

In Eukarya and Archaea, PCNA-binding proteins generally interact with PCNA via a conserved PIP box motif (e.g. archaeal DNA polymerase B and flap endonuclease 1 have a typical PIP box motif at their C termini) (reviewed in Ref. 7). Human Lig I has a typical PIP box at the N-terminal tail, but PfuLig lacks a long N-terminal tail. We identified the PCNA-interacting motif of PfuLig in a loop structure that connects two {alpha}-helices in the N-terminal DBD. Based on the information from the crystal structure of human Lig I (N-terminally truncated mutant) complexed with a nicked duplex DNA, a model structure of the DNA ligase·PCNA complex with 1:1 stoichiometry was proposed (14). This interaction is likely to involve "face to face" binding because of the proteins' similar sizes and the toroidal structure of PCNA. After binding to the DNA·PCNA complex via the PCNA-binding motif in the DBD, the conformations of the CD may change freely to encircle a nicked DNA because it has no interactive region with PCNA (Fig. 4), and subsequently, the enzyme catalyzes the nick-joining reaction.

PCNA Is a Scaffold Protein for Binding to DNA under Physiological Ionic Conditions—There have been some contradictory observations about the stimulatory effect of human PCNA on Lig I activity. One group suggested that these discrepancies are due to differences in experimental conditions (16). As shown in Fig. 3 (B and C), the PfuLig activity was inhibited by PCNA at low salt concentrations (0–0.05 M KCl and 0–0.1 M potassium glutamate), but the activity was enhanced in a salt concentration-dependent manner at >0.05 M and peaked at 0.2 M for KCl and at >0.1 M and peaked at 0.3–0.4 M for potassium glutamate. In a previous report on the inhibitory effect of PCNA on human Lig I, the inhibitory effect was observed at 0 and 50 mM NaCl, and no effect of PCNA on ligation was observed at 100 mM NaCl (in pH 6.5 buffer) (15). Based on our findings, the stimulatory effect may be observed at 150 mM NaCl, which is near the physiological ionic strength within human cells. However, it is not easy to discuss the differences in the assay conditions because the salt concentration of each fraction containing the purified recombinant protein is not always obvious from the information presented.

It can be predicted that, by themselves, eukaryotic DNA ligases cannot bind to DNA to catalyze the nick-joining reaction at a physiological salt concentration, but they can recognize the substrate DNA by interacting with PCNA on a nicked DNA. Most of the DNA modification enzymes can interact with substrate DNAs to express their function at low salt concentrations, but lose their activities at high salt concentrations in vitro. The DNA binding abilities of these enzymes themselves are probably inhibited by salt in the cells. Each protein involved in DNA replication and repair has to work at a certain time in the successive processes at the appropriate site. To control the specific timing and the position for each related protein factor to access the target DNA in vivo, the salt concentration, which prevents nonspecific binding of protein factors in the cells, is especially important, and in the case of replication fork progression, for example, PCNA probably functions as a platform to control the order and the sites of interacting proteins involved in this successive reaction process.

Conserved Residues in the Novel PCNA-binding Motif (QKSFF)—The well known PIP box is generally located in the N- or C-terminal region within the peptide chain of PCNA-interacting proteins. However, the PCNA-binding motif (QKSFF) found in this study is in the middle of the PfuLig protein. This novel PCNA-binding motif resembles a putative bacterial beta-clamp-binding motif (QL(S/D)LF) that is located not only at the terminus but also in the middle of some beta-clamp-interacting proteins. In this bacterial motif, the pair of hydrophobic residues (LF) is important for binding to the beta-clamp (10). In PfuLig, the corresponding hydrophobic residues (FF) are also important for binding to PCNA, and furthermore, our work has shown that the former residue (Phe106) is more critical than Phe107 (Fig. 6C). Moreover, the importance of Gln103 was revealed in our experiments. A structural comparison of the novel motif in the PfuLig crystal with the PIP box motif in the RFC large subunit in the cocrystal with PfuPCNA (36) is shown in Fig. 7. Interestingly, the locations of the amino acid residues responsible for the hydrophobic and ionic interactions clearly correspond to each other, and especially the positions of Gln470 and Phe476 in the PIP box of the RFC large subunit, corresponding to Gln470 and Phe476 in PfuLig, are remarkably conserved among the PIP box sequences. This new motif may represent a shorter version of the original PIP box. To determine the detailed role of each amino acid in PCNA binding, an x-ray crystallographic structure of the PfuLig·PfuPCNA complex will be required.

Interestingly, this novel PIP box motif is widely conserved in the same region of other archaeal DNA ligases (Fig. 8A). The QKSFF sequence is completely conserved, especially in Thermococcales (Pyrococcus and Thermococcus species). In addition, the basic residues located in the region upstream of the motif are also conserved, and especially the remarkable cluster of basic residues is conserved in the DNA ligases from Thermococcales and some methanogens. As we proposed previously based on mutational analyses of the RFC large subunit from P. furiosus (37), these basic residues may function in the formation of the stable ligase·PCNA·DNA ternary complex in these organisms.

We examined the sequences of the eukaryotic DNA ligases and found that they also have the Archaea-type PIP box in the middle of the peptide chain (Fig. 8B). Eukaryotic Lig I may bind to PCNA at the site corresponding to the motif that we found in this study as discussed above, in addition to the N-terminal PIP box. These analyses showed that the DNA ligase-PCNA interaction mode is also interesting from an evolutionary perspective, and we plan to investigate this possibility by introducing mutations at the conserved Glu residue in the human Lig I protein.


   FOOTNOTES
 
* This work was supported in part by the Japan Science and Technology Agency and by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to Y. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 Supported by the Human Frontier Science Program and the Hou-ansha Foundation. To whom correspondence should be addressed. Tel.: 81-92-642-4217; Fax: 81-92-642-3051; E-mail: ishino{at}agr.kyushu-u.ac.jp.

2 The abbreviations used are: Lig I, DNA ligase I; PCNA, proliferating cell nuclear antigen; PIP, PCNA-interacting protein; DBD, DNA-binding domain; PfuPCNA, P. furiosus PCNA; PfuLig, P. furiosus DNA ligase; DTT, dithiothreitol; CD, catalytic domain; SPR, surface plasmon resonance; RFC, replication factor C. Back

3 Nishida, H., Kiyonari, S., Ishino, Y., and Morikawa, K. (2006) J. Mol. Biol. 360, 956–967. Back

4 Sequences are available upon request. Back



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