Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan
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Abstract |
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Keywords: -like DNA polymerase/Archaea/DNA replication/hyperthermophile/site-specific mutagenesis
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Introduction |
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Studies on the structurefunction relationships of DNA polymerases in family B have generated great interest due to the fact that the catalytic subunits of the replicative enzymes in eukaryotes (Pol , Pol
and Pol
) belong to family B. Mutational analyses have been done for human Pol
(reviewed in Copeland et al., 1995),
29 Pol (reviewed in Blanco and Salas, 1995, 1996), T4 Pol (reviewed in Reha-Krantz, 1995) and Escherichia coli Pol II (Ishino et al., 1994
). The three-dimensional structure of a family B DNA polymerase was not determined until recently. The crystal structure of the family B DNA polymerase from bacteriophage RB69 was solved two years ago (Wang et al., 1997
), and more recently, the structure of a family B DNA polymerase from the hyperthermophilic archaeon, Thermococcus gorgonarius, was published (Hopfner et al., 1999
). These results showed that both proteins are ring-shaped with a small hole in the center in which the single polypeptide chain is folded into five structurally and functionally distinct domains. The catalytic palm domain of the family B DNA polymerases have the same topology as found in other polymerases, including different categories, such as reverse transcriptase and RNA polymerases; however, the thumb and finger domains are apparently unrelated to other known polymerases.
To expand our knowledge of the structurefunction relationships of the family B DNA polymerases, and especially to understand the structural relationship between the DNA polymerizing and 3'5' exonucleolytic activities in the polymerase protein, we prepared several mutant proteins of Pol BI from P.furiosus by a unidirectional deletion strategy and site-specific mutagenesis, and analyzed their activities.
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Materials and methods |
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Enzymes and kits for in vitro manipulations of DNA and synthesized oligonucleotides were purchased from Takara Shuzo (Kyoto, Japan). The [methyl-3H]TTP and [32-P]dCTP were purchased from Amersham (Buckinghamshire, UK) and the [
32-P]ATP was purchased from NEN Life Science Products (Boston, MA).
Construction of the P.furiosus polA gene expression plasmid
To adjust the initiation codon of the polA gene to the NcoI site within the initiation codon of the lacZ gene in pTV119N (Takara Shuzo), PCR cloning was done using a primer containing a -CCATGG- sequence at the initiation codon, and for this reason, the second codon was changed from Ile (ATT) to Val (GTT). The resultant plasmid was designated pFuNSPN. For further improvement of expression, the structural gene was excised from pFuNSPN by NcoISphI digestions and was inserted into the corresponding sites of pSTOP (described below). The resultant plasmid was designated pFU3.
Preparation of mutant polA genes
To make a C-terminal truncated Pol BI, the pSTOP plasmid, with three stop codons in tandem in the three different translational frames, was constructed. The two oligonucleotides, 5'-AGTTAGTTAATCGAT-3' and 5'-TCGATCAATCAATTAGCTATTAA-3', were annealed and inserted into the SacIEcoRI site of pTV119N. The plasmid pFU3, with the structural gene of polA in pSTOP, was subjected to the unidirectional deletion procedure using the kilosequence deletion kit (Takara Shuzo). For the initial digestion of the plasmid, HincII and KpnI were used to create blunt-ended and 3'-protruded termini, respectively. For site-specific mutagenesis, a PCR-based method was utilized. PCR primers D405A-F, 5'-ACATAGTATACCTCGCGTTTAGAGCCCT-3' and D40-5E-F, 5'-ACATAGTATACCTCGAGTTTAGAGCCCT-3', include AccI (-CTCGCG-) and XhoI (-CTCGAG-) recognition sites, respectively, to convert the codon for D405 (GAT) to A (GCG) or E (GAG). Using pFU3 as a template, PCR was performed using the combination of D405A-F and a universal primer, which binds to the proximal site of the multicloning site of the vector plasmid. In parallel, the reaction using D405A-R (complementary to D405-F) and a universal RV-N was done. PCR products were digested with AccIXbaI (for the former) or AccIEcoRI (for the latter) and were ligated to EcoRIXbaI-digested pFU3. The conversion of D405 to E was done in the same way, but with XhoI instead of AccI. The nucleotide sequences of the prepared plasmids were confirmed by using a fluorescent DNA sequencer (ABI377A, PE Applied Biosystems, Foster city, CA).
Purification of wild-type and mutant Pol BI proteins
Escherichia coli JM109/pFU3 was grown at 37°C with shaking in 1 l of L-broth containing 50 mg ampicillin. When the culture reached 0.6 A600, isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the culture was incubated for a further 14 h. The cells were harvested and disrupted by sonication in buffer A, containing 150 mM TrisHCl, pH 7.6, 2 mM EDTA, 2.4 mM phenylmethanesulfonyl fluoride (PMSF) and 0.2 % Tween 20. The supernatant was incubated at 80°C for 15 min to denature and precipitate most of the E.coli proteins. The heat-treated supernatant was dialyzed against buffer A containing 10% glycerol, and then was subjected to anion-exchange chromatography (RESOURCE Q, Amersham Pharmacia), which was developed with a linear gradient of 0500 mM NaCl. The fractions that were eluted at around 100 mM NaCl, containing the Pol BI protein, were subjected to cation-exchange chromatography (RESOURCE S, Amersham Pharmacia) on a column equilibrated with the same buffer. The Pol BI protein was eluted with 150200 mM NaCl in the linear gradient. The mutant proteins were purified by the same procedure, except for two proteins, 2 (
Leu717Ser775) and
1 (
His672Ser775), which did not stick to the RESOURCE S column and were recovered from the flow-through fraction.
Assay of DNA polymerizing activity
The DNA polymerizing activity was assayed by measuring the incorporation of [methyl-3H]TTP into acid insoluble materials, as described previously (Uemori et al., 1995).
Assay of exonucleolytic activity
The 5'-protruding end of the BamHI-digested pUC118 was filled by the Klenow polymerase reaction with dNTPs including [-32P]dCTP. Alternatively, a deoxyoligonucleotide of 40 bases in length (5'-dTACAGAAGATGGGAGGAGGGACCGGACTCAACTTCTCAAA-3') was labeled with polynucleotide kinase (PNK) with [
-32P]ATP and was used directly as the single-stranded substrate or after annealing with its complementary strand for the double-stranded substrate. The reaction mixture, containing 20 mM TrisHCl, pH 8.0, 2 mM MgCl2, 10 mM KCl, 6 mM (NH4)2SO4, 1% Triton X-100, 100 µg/ml BSA, 1pmol substrate DNA, and 2µg Pol BI in 20µl, was incubated at 74°C (for the plasmid substrate) or 56°C (for the oligonucleotide substrate), and then the radioactivity in the ethanol-soluble fraction was measured by Cerenkov radiation or the reaction mixture was subjected to electrophoresis on 8% polyacrylamide gel containing 8 M urea.
Western blot analysis
Purified proteins were separated by 7.5% SDSPAGE followed by electroblotting onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA). The blots were processed with an enhanced chemiluminescence system (Amersham) as described (Cann et al., 1998).
Activity-gel analysis
The analysis of the DNA polymerizing activity in situ, to determine the catalytically active polypeptide, was carried out basically as described earlier (Wernette and Kaguni, 1986). The conditions were the same as those described in our previous report (Imamura et al., 1995
).
Gel-retardation assay
Gel-retardation assays were carried out using 32P-labeled oligonucleotides as probes. The 17mer oligonucleotide (5'-dAGCTATGACCATGATTA-3') was labeled by polynucleotide kinase (PNK) with [-32P]ATP and was used directly as the single-stranded probe or after annealing with the other 49mer oligonucleotide (5'-dAGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCAT GGTCATAGCT-3') including the complementary sequence for a templateprimer structure. The reaction mixtures for proteinDNA binding (10 µl) contained 20 mM Trisacetate, pH 8.0, 10 mM MgCl2, 1 mM DTT, 100 µg/ml BSA, 5% glycerol, 0.5 pmol DNA (a mixture of the single-stranded and annealed primers) and various amounts of the proteins (0.0510 pmol) were incubated at 60°C for 5 min. Electrophoresis was carried out in 1% agarose gels, containing 4 mM Trisacetate pH 8.0 and 0.1 mM EDTA, which were run at 50 V in the same buffer at room temperature. After electrophoresis, the gels were dried and autoradiographed.
Polymerase/exonuclease coupled assay
The coupled assay was carried out basically as described previously (Truniger et al., 1996). The template (d49mer)-primer (d17mer) used for the gel-retardation assay was used as the substrate. The reaction mixture (20 µl) contained 20 mM TrisHCl, 2 mM MgCl2, 10 mM KCl, 6 mM (NH4)2SO4, 0.1% Triton X-100, 0.5 pmol templateprimer (32P-labeled at the 5'-terminus of the primer), 1 µg enzyme and various amounts (0100 µM) of dNTPs. After an incubation for 5 min at 60°C (a lower than optimal temperature for consideration of the templateprimer stability), the reaction products were analyzed by electrophoresis on an 8% polyacrylamide gel containing 8 M urea.
Structure drawing
The three-dimensional structure of a family B DNA polymerase of T.gorgonarius was retrieved from the Protein Data Bank (PDB ID code 1TGO), and the figure was drawn with the QUANTA 97 program (Molecular Simulations Inc., San Diego, CA).
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Results and discussion |
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To increase expression of the Pol BI protein in E.coli over that from our previous study (Uemori et al., 1993), the initiation codon of polA was adjusted to that of the lacZ gene in plasmid pTV119N, and the resultant plasmid was designated pFuNSPN. The thermostable DNA polymerizing activity produced in E.coli JM109 carrying pFuNSPN was 29.5 U/ml culture, which was obtained from the cell extracts after heating at 75°C for 15 min to inactivate all of the DNA polymerizing activities from E.coli. The DNA fragment inserted into the plasmid pFuNSPN contains an additional 1.1 kb downstream from the polA gene, and therefore, the 2.5 kb spanning only the structural gene of polA was excised and inserted again into the same type of expression vector. Escherichia coli JM109 carrying the resultant plasmid, pFU3, produced 125 U/ml culture of DNA polymerizing activity, which was fivefold higher than that from JM109/pPuNSPN. The changes in the codon usage at the translational initiation region of polA gene to the optimal codon usage for E.coli were expected to enhance the gene expression, as in the case of Thermus aquaticus (Taq) DNA polymerase in E.coli (Ishino et al., 1994
). However, several trials were not effective (data not shown).
Purification of recombinant Pol BI
Starting from the E.coli transformant (JM109/pFU3), a simple and rapid purification procedure was established, which leads easily to homogeneous P.furiosus Pol BI in good yield. After heat-treatment of the crude cell extract of the E.coli transformant, two sequential chromatographic steps were sufficient to obtain the purified Pol BI protein. The purity of the Pol BI in each fraction was checked by SDSPAGE (Figure 1). About 2 mg of the purified Pol BI were obtained from 1 l culture. This result is comparable to that reported for the expression of the polA gene using the pET-expression system in E.coli (Lu and Erickson, 1997
).
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To investigate the structural relationship between the two activities in Pol BI, the exonuclease and the DNA polymerase, nested deletions from the C-terminus of the protein were designed. The mutants obtained from this strategy were 1,
2 and
3, which lack 30, 59 and 104 amino acids, respectively, from the C-terminus of Pol BI (Figure 2A
). Furthermore, to determine whether it would be possible to prepare a mutant protein that has only the exonuclease activity, the substitution of a candidate residue within the polymerizing active site was carried out. Blasco et al. (1993) reported that the D249E mutant of
29 DNA polymerase lost most of the DNA polymerizing activity, but retained its 3'
5' exonuclease activity. Therefore, substitutions of the corresponding Asp (D405A and D405E) of Pol BI were tried (Figure 2A
). The preparation of the mutant genes and their expression are described in the Materials and methods. The purification behaviors of
1, D405A and D405E were exactly the same as that of the wild-type protein. However,
2 and
3 did not bind to the cation-exchange column under the conditions in which the others bound, which suggests that some distinct conformational changes in
2 and
3 occurred as a result of the C-terminal deletions. An SDSPAGE analysis of the purified mutant proteins as well as the wild-type proteins is shown in Figure 2B
. In the case of
2 and
3, smaller bands were detected in addition to the bands with appropriate sizes (lanes 5 and 6). Judging from a western blot analysis (Figure 2C
), the smaller bands are derived from degradation of the mutant proteins. The conformational change of the thumb domain may cause them to be sensitive to some protease.
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Using the purified proteins, the effects of each mutation on the DNA polymerizing and 3'5' exonucleolytic activities were investigated. As shown in Table I
, the specific activities for DNA polymerization, calculated from the incorporation of [3H]TTP into DNaseI-activated calf thymus DNA, were varied. To visualize the DNA polymerizing activities of the mutant proteins, an activity-gel analysis was done. As shown in Figure 3
, the bands of the wild-type and
1 proteins, but not those of the others, incorporated the [32P]CMP as a substrate into the DNA strands. This result corresponded to the specific activities of each mutant protein shown in Table I
. The signal of
1 (lane 5) looks stronger than that of the wild-type (lane 2), as shown by the difference in their specific activities. As predicted, the substitution of Asp405 drastically affected the activity; especially, the D405A mutation decreased the polymerase activity by 440-fold. Asp405 can be aligned to one of the two carboxylates, which are absolutely conserved among all of the polymerases excluding the terminal transferase family (Delarue et al., 1990
). Our results with the Asp405 mutations further support the idea that this Asp participates in the catalysis of the DNA polymerizing reaction. On the other hand, the truncation analysis showed that only
1 has activity comparable with that of the wild-type, but
2 and
3 are 32- and 122-fold less active, respectively. These results indicate that at least 32 residues from the C-terminus of Pol BI are not necessary, and there may be a critical position for maintaining the DNA polymerizing activity within residues 3059 from the C-terminus.
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Using equal amounts of mutant protein, the 3'5' exonucleolytic activities were measured. An exonuclease assay, using linearized pUC118 labeled at the 3'-terminus with 32P, was performed, and the released 32P was counted. The relative activities of the mutants were compared with that of the wild-type protein, as shown in Table I
. All five of the mutations affected the exonucleolytic activity. The two substitutions and
1 were decreased to 10% and the two other deletions were decreased to 3% of the wild-type Pol BI activity. To visualize the activities, another assay using a 32P-labeled oligonucleotide with or without annealing to the complementary strand was done, and the reaction products were analyzed by gel electrophoresis followed by autoradiography (Figure 4
). The activities of the mutant proteins relative to the wild-type were comparable to those found in the above experiment in Table I
. The single-stranded DNA substrate was more sensitive than the double-stranded DNA in all cases.
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To investigate the effect of the mutations on the synthesis/degradation balance, a pol/exo coupled assay was performed. As shown in Figure 5, for wild-type Pol BI, most of the primers were extended when the dNTP substrates were provided to 1 µM, and degradation was observed in the reaction with 100 nM dNTPs. In the case of the
1 mutant protein, the primers were extended even with 10 nM dNTPs. This result is consistent with the finding that the
1 mutant has higher polymerizing and much lower exonucleolytic activities as compared with those of the wild-type Pol BI, as shown above. A distinct amount of extended products synthesized by D405E was observed in the presence of 10 µM dNTPs. No extension and the same level of primer degradation were observed with D405A and various concentrations of dNTPs. These results show that Asp405 is very important for the affinity of the polymerase active site in Pol BI to the primer-terminus, and the substitution of this residue shifts the balance of the affinities of the two active sites towards the exonuclease site. Neither extension nor degradation was observed with the other two mutant proteins,
2 and
3 (data not shown).
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Stable interaction of the wild-type and mutant Pol BI proteins with a templateprimer structure was investigated. Using a mixture of double-stranded templateprimer and the single-stranded primer as probes, gel retardation assays were performed. As shown in Figure 6, the wild-type, D405A and D405E proteins gave rise to a single retardation band which possibly corresponds to a proteinDNA complex both in the presence and absence of MgCl2. Judging from the electrophoresis profiles, the templateprimer is preferentially bound as compared with the single-stranded primer in all cases. In the presence of MgCl2, the shifted band became weaker in proportion to the amount of wild-type Pol BI, which is due to the degradation of the DNA by its exonuclease activity. The shifted band remained and became stronger with higher amounts of protein in the absence of MgCl2. The
1 mutant protein has strong DNA polymerizing activity, but the shifted band was observed only with very faint intensity in both the presence and absence of MgCl2. No retardation was observed with the
2 and
3 mutants (data not shown). These results suggest that the retarded band observed in the gel electrophoresis is the complex in which the templateprimer is bound at the exonuclease active site, but not at the polymerase active site. A shifted band with different mobility from that observed in others appeared by
1 after a long exposed autoradiography (Figure 6
), which may be the complex bound at the polymerase site, and the complex may be less stable than that detected clearly under the gel-retardation assay conditions described here. The thumb subdomain is probably very important for the stable binding of Pol BI to the templateprimer with the exonuclease mode.
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The present results support the idea that there is a functional interaction between the exonuclease and polymerase sites of P.furiosus Pol BI. There have been several reports that suggest the interdependence between the polymerase and exonuclease domains in the family B DNA polymerases. Single point mutations in the exo or pol domains of the Herpes simplex virus (HSV) DNA polymerase (Gibbs et al., 1991; Wang et al., 1992
), the
29 DNA polymerase (Soengas et al., 1992
) and the T4 DNA polymerase (Reha-Krantz et al., 1993
; Sattar et al., 1996
) affected both activities.
On the other hand, an archaeal family B DNA polymerase from Sulfolobus solfataricus has been demonstrated to have modular organization of its catalytic activities, in which 3'5' exonucleolytic and polymerizing activities can be divided into the two polypeptides (Pisani and Rossi, 1994
). The crystal structure of a family B DNA polymerase from the bacteriophage RB69 (Wang et al., 1997
) and an archaeon, T.gorgonarius (Hopfner et al., 1999
) have recently been solved. These two structures consist of the five distinct structural domains: N-terminal, 3'
5' exo, palm, finger and thumb. The sequence comparison showed that the P.furiosus Pol BI is 95% similar (80% identical) to the T.gorgonarius polymerase, and therefore it is possible to superimpose the structure of the P.furiosusPol BI onto the published structure of the T.gorgonarius Pol. Figure 7
shows the three-dimensional structure of the T.gorgonarius DNA polymerase. The thumb domain looks closely related to the exonuclease domain (colored in yellowish green). The corresponding regions of the P.furiosus Pol BI,
1,
1
2 and
2
3, that were deleted in this study are shown in yellow, blue and red, respectively. The proper conformation of the thumb domain seems to be necessary for both the exonuclease and polymerase activities. The most C-terminal region, shown in yellow, does not contact the exonuclease domain directly; however, the exonuclease activity of
1 decreased to 10% of that of the wild-type. The deletion of this region may affect the stability of the thumb domain, thus perturbing its interaction with the exonuclease domain, and preventing the proper interaction with the palm and finger domains that is necessary for the proper binding to the DNA for the exonucleolytic activity, even though the deletion does not affect the DNA polymerizing activity. The deletions in
2 and
3 proteins almost disrupted the thumb subdomain, and these mutant proteins lost both activities. It is reported that a 75 residue subdomain (665729) in the thumb domain of T.gorgonarius Pol fixes the exonuclease domain and contributes to the editing channel (Hopfner et al., 1999
). This subdomain corresponds to the region including the entire
2
3 and the half of
1
2 of P.furiosus Pol BI. Therefore, destabilization of the templateprimer binding by the
2 and
3 mutant proteins possibly affects mainly both polymerizing and exonucleolytic activities, as supported by the results of the gel-retardation assays.
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In conclusion, our mutational analysis further supports the idea that the polymerase and exonuclease domains in the family B DNA polymerases are functionally interdependent. More detailed analyses will be necessary to understand the molecular mechanism of the functional interaction between the two activities in the DNA polymerases.
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Acknowledgments |
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Notes |
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References |
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Received August 26, 1999; revised October 14, 1999; accepted October 25, 1999.