DNA Polimerasa Eq-5

7/26/2019 DNA Polimerasa Eq-5

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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

A new DNA polymerase I from Geobacillus caldoxylosilyticus

TK4: cloning, characterization, and mutational analysis

of two aromatic residues

Cemal Sandalli &Kamalendra Singh &

Mukund J. Modak &Amit Ketkar &Sabriye Canakci &

smail Demir &Ali Osman Belduz

Received: 8 January 2009 /Revised: 11 March 2009 / Accepted: 11 March 2009 /Published online: 14 April 2009# Springer-Verlag 2009

Abstract DNA polymerase I gene was cloned andsequenced from the thermophilic bacterium Geobacilluscaldoxylosilyticus TK4. The gene is 2,634 bp long andencodes a protein of 878 amino acids in length. Theenzyme has a molecular mass of 99 kDa and showssequence homology with DNA polymerase I from Bacillusspecies (89% identity). The gene was overexpressed in

Escherichia coliand the purified enzyme was biochemical-ly characterized. It has all of the primary structural elementsnecessary for DNA polymerase and 53 exonucleaseactivity, but lacks the motifs required for 3 5 exonucle-ase activity. 5 nuclease and 35 exonuclease assays

confirmed that Gca polymerase I has a double-strandedDNA-dependent 53 nuclease activity but no 35exonuclease activity. Its specific activity was observed to be495,000 U/mg protein, and KD

DNA, KDdNTP, and Kpolwere

found to be 0.19 nM, 22.64 M, and 24.99 nucleotides1,respectively. The enzyme showed significant reverse-transcriptase activity (RT) with Mn2+, but very little RTactivity with Mg2+. Its error rate was found to be 2.5105

which is comparable to that of the previously reported errorrate for the E. coli DNA polymerase I. Two aromaticresidues required for dideoxyribonucleotide triphosphatesensitivity (F712Y) and strand displacement activity(Y721F) were identified.

Keywords Geobacillus caldoxylosilyticus TK4 .

DNA polymerase I . Thermophilic . Mutation

Introduction

The integrity of all organisms depends on faithful genomereplication. This is accomplished by DNA polymeraseswhich synthesize DNA with extraordinary fidelity andefficiency to ensure proper transfer of genetic informationfrom parent to progeny. For this purpose, all free-livingorganisms possess more than one kind of DNA poly-merases equipped with different functions. Based uponsequence comparison, DNA polymerases have been classi-fied into six families: A, B, C, D, X, and Y. The DNA

polymerases that share sequence homology with Escher-ichia coliDNA polymerase I, II, and III have been assignedto the A, B, and C families, respectively. The DNA

polymerases which did not show a sequence conservationwith E. coli pol I, II, and III were grouped in X family(Delarue et al.1990). Thus, mammalian DNA polymeraseB belongs to the X family. Family D polymerases arethought to be replicative polymerases, and all knownexamples are found in Euryarchaeota of Archaea (Ishinoet al.1998). The newest class of DNA polymerases is the Yfamily which contains the DNA polymerases capable oflesion-bypass DNA synthesis (Ohmori et al.2001; Hubscheret al.2002).

Appl Microbiol Biotechnol (2009) 84:105117

DOI 10.1007/s00253-009-1962-3

C. SandalliDepartment of Biology, Faculty of Arts & Sciences,

Rize University,53100 Rize, Turkey

K. Singh :M. J. Modak : A. KetkarDepartment of Biochemistry and Molecular Biology,UMDNew Jersey Medical School,

Newark, NJ, USA

S. Canakci :. Demir: A. O. Belduz (*)Department of Biology, Faculty of Arts & Sciences,Karadeniz Technical University,6180 Trabzon, Turkeye-mail: [email protected]


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The A family DNA polymerases (pol Is) from differenteubacteria share significant sequence homology and,therefore, are structurally and functionally similar to eachother. A typical pol I protein consists of a single poly-

peptide chain comprising an N-terminal domain with a 53 exonuclease activity, a central domain with a 35exonuclease activity (or proofreading) and a C-terminal

domain with DNA polymerase activity. In some DNA polIs, like bacteriophage T5 and T7 DNA polymerases, the N-terminal domain which contains 5 nuclease activity isfound as separate polypeptides. The proofreading domainof other members of DNA polymerase I class such asThermus aquaticus (Taq pol I; Lawyer et al. 1989),

Mycobacterium tuberculosis (Mtb pol I; Arrigo et al.2002), Streptococcus pneumoniae (Spn pol I; Amblar etal. 1998), Bacillus caldotenax (Bca pol I; Uemori et al.1993), and Bacillus stearothermophilus (Bstpol; Aliotta etal.1996) is inactive. The pol Is from different species alsoexhibit differences in other biochemical properties like

specific activity, dideoxyribonucleotide triphosphate(ddNTP) sensitivity, strand displacement synthesis, andRNA-dependent DNA synthesis.

The A family of DNA polymerases contains severalmembers that have been extensively used as tools invarious gene technology procedures, such as probe label-ing, DNA sequencing, and polymerase chain reaction(PCR). There are also examples of engineering of the

polymerase domain to improve the incorporation ofdideoxynucleotides and other nucleotide analogues (Taborand Richardson 1995; Li et al. 1999). The first BacillusDNA polymerase I was isolated and cloned from B.

stearothermophilus and it was biochemically characterized(Mead et al. 1991; Lu et al. 1991; Phang et al. 1995).Similar enzymes have been isolated from other bacilli, e.g.,

B. caldotenax (Uemori et al. 1993). These enzymes differslightly in their characteristics. However, the biochemical

properties of thermostable DNA polymerases from otherBacillus species (Geobacillus, Oceanobacillus, Anoxyba-cillus, etc.) are still unknown. There is only one previousstudy which reported the cloning of a DNA polymerasefrom the genus Geobacillus (Khalaj-Kondori et al. 2007).In this study, we characterized a new DNA polymerase Ifrom Geobacillus caldoxylosilyticus TK4 strain isolatedfrom a hot spring in Turkey (Canakci et al. 2007).

Materials and methods

Microorga nism and determination of the polA gene

sequence The strain TK4 of G. caldoxylolyticus wasisolated from the Kestanbol hot spring in Turkey anddeposited in the National Collections of Industrial Food andMarine Bacteria under the number 14283 (Canakci et al.

2007). We designed two degenerate primers (Deg-PolF: 5-CCBAAYYTSCARAACATHCC-3 and Deg-PolR2: 5-KASNAKYTCRTCRTGNAC YTG-31) for the conservedregion in family A DNA polymerases (Uemori et al 1993).By using these primers and genomic DNA from Gca TK4,a product of approximately 0.6 kb was amplified in PCR.The fragment was cloned into pGEM-T easy vector

(Promega) and sequenced (Macrogen, South Korea). Thecomplete coding sequence of Gca pol I was obtained byusing a second degenerate primer (Pol_F: 5-ATGAGRTTRAARAAAAARCTM-3) hybridized to start site of thegene and by performing an inverse PCR (iTK4-F: 5-CGGCGATGAATACGCCGATTC-3 and iTK4-R: 5-CGTGGCGAAACGCATCGAT-3) using this known siteto determine 3 end of the gene with ClaI restrictiondigestion. The PCR was performed at 94C for 3 min and94C for 30 s, 52C for 1 min, and 72C for 2 min for30 cycles. We combined the sequences to have thecomplete sequence of the polymerase I gene ofGca TK4.

Cloning of Gca pol I into expression vector and site-

directed mutagenesis Gcapol I gene was amplified by twospecific primers (TK4F: 5-GACCATGGGGTTGAAAAAA AAGCTAGTATTTG-3 and TK4R: 5-CAGGATCCTTACTTTGCGTCATACCATGT TGG-3) that have NcoIand BamHI restriction sites (underlined) to allow in-frameligation into the pET-15b expression vector (Novagene).PCR was performed using 2.5 U of Expand High FidelityTaqDNA polymerase (Fermentas) in a 50-l PCR reactionmixture containing 0.2 mM of each dNTP, 300 ng of each

primer, and 100 ng Gca TK4 genomic DNA. The PCR

cycle was as follows: 95C for 5 min, 30 cycles of 94C for1 min, 58C for 1 min, 72C for 4 min, and final extensionat 72C for 7 min. A predicted 2.63-kb DNA fragmentobtained from the PCR was digested with NcoI andBamHIand ligated into the pET-15b expression vector digestedwith the same enzymes. The resulting recombinant plasmidnamed pET15b-GcaDNA pol was transformed into E. coliBL21 (DE3)pLysS for overexpression. We used a PCR-

based protocol as described in Stratagenes QuickChangesite-directed mutagenesis kit to generate the desired muta-tions in polAgene ofGca TK4 using the primers Y721F-F:5-CGGCATCAGCGACTTCGGG CTGTCAC3 andY721F-R: 5-GTGACAGCCCGAAGTCGCTGATGCCG-3 for Y721F mutation and F712Y-F: 5-CGAAAGCGGTG AACTATGGCATTGTTTACGG-3 and F712Y-R: 5-CCGTAAACAATGCCATAGTTCACCGCTTTCG-3 for F712Ymutation. While the Phe712 residue inGca pol I was changedto Tyr712 to improve its ddNTP sensitivity, Tyr721 residue waschanged to Phe721 to see its effect on strand displacementsynthesis activity. The plasmid containing the pET15b-GcaDNApol was used for generating mutant derivatives, and allmutations were confirmed by DNA sequencing of plasmid.

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Overexpression and purification of proteins E. coli cellsharboring Gca pol I on pET-15b were grown to an opticaldensity at 600 nm of about 0.6 in LB medium containing50 g/ml ampicillin at 37C, and expression was induced

by the addition of 1 mM isopropyl -D-thiogalactopyrano-side. After induction for 3 h at 37C, the cells wereharvested by centrifugation. The recombinantGca pol I and

its mutant derivatives (Y721F and F712Y) were purified byperforming heat treatment, DEAE-cellulose (Uemori et al.1993), and BioRex 70 cationic chromatography (David etal.1990). The expression and purification ofMtb DNA polI enzyme was done according to Arrigo et al (2002). Theexpression (Polesky et al.1990) and purification ofE. coli

pol I, KF, and KF exo were done according to Joyce andDerbyshire (1995). Purification of the enzyme was moni-tored by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE; 8% gel) at every step. Proteinconcentrations were determined by the NanoDrop (ND-1000 Spectrophotometer) method.

5 end and 3 end labeling of the substrates The oligonu-cleotides 14-mer, 33-mer, and 18-mer listed in Table1were

phosphorylated at the 5 end by T4 polynucleotide kinaseand [-32P]ATP). The oligonucleotide 42-mer was labeledat the 3 end by KF and [-32P]ATP. The radiolabeledoligonucleotides were purified by electrophoresis on a 20%acrylamide7-M urea gel as described by Maxam andGilbert (1980) and desalted on a NAP 10 column(Pharmacia). These syntetic strands were annealed to theircorresponding template at a 5:1 (template/primer) ratio asshown in Table1.

Optimum temperature and thermal stability To determinethe optimum temperature ofGca pol I, enzymatic reactionsat various temperatures over the range 3085C were

performed in a reaction mixture (50 l) containingpolymerase activity buffer (containing 50 mM TrisHCl,pH 8, 2 mM dithiothreitol, 0.1 g/ml bovine serumalbumin), 6 mM MgCl2, 2 M of 33/16-mer DNA(Table 1), 50 M of each dNTP, and 3H-dTTP (0.5 Ci/assay). Reactions were initiated by the addition of enzyme,incubated at each temperature for 5 min, and then quenchedwith ice-cold 5% trichloroacetic acid containing 10 mMPPi. Acid-precipitable material was collected on Whatmanglass fiber filter and the radioactivity was measured byscintillation spectroscopy. For thermal stability, aliquots(10 l) of Gca pol I in plastic tubes were incubated atvarious temperatures over the range 5085C for 5 min andthen chilled on ice. These heat-treated enzymes were testedfor residual DNA polymerase activity by primer extensionassay in a reaction mixture (6 l) containing polymeraseactivity buffer, 6 mM MgCl2, 100 ng 32/14-mer DNA(Table 1), and 50 M dNTP at 60C for 30 s and 2 and

5 min. The reactions were terminated by the addition of 2stop solution (20 mM ethylenediamine tetraacetic acid(EDTA), 0.2% (w/v) SDS, 80% (v/v) formamide, and0.008% (w/v) each of bromophenol blue and xylene cyanol)and heated at 95C for 5 min. A 6-l sample was loadedonto a 16% polyacrylamide8-M urea gel, and the productswere separated at 1,500 V for 3 h. The resulting gel was

dried and exposed to a phosphor screen that was thenanalyzed on a Typhoon 9400 (Amersham Biosciences).

Optimum pH and divalent ion concentrations To determinethe optimum pH of Gca pol I, enzymatic reactions atvarious pHs from 7.2 to 10.2 were performed in a reactionmixture (50 l) containing polymerase activity buffer,6 mM MgCl2, 2 M 33p16/16-mer DNA, 50 M of eachdNTP, and 3H-dTTP (0.5 Ci/assay). Reactions wereinitiated by the addition of enzyme, incubated at 60C for5 min, and then quenched and measured as described in the

previous section. Optimum divalent ion concentration was

determined similarly as in polymerase activity assay, usingdifferent concentrations of Mg+2 and Mn+2 from 1 to12 mM and from 0.5 to 10 mM, respectively.

Determination of DNA polymerase activity Specific en-zyme activity of Gca pol was performed in a reactionmixture (50 l) containing polymerase activity buffer,6 mM MgCl2, 2 M 33p16/16-mer DNA, 50 M of eachdNTP, 3H-dTTP (0.5 Ci/assay), and 60 to 600 pg Gca polI at 60C for 10 min. The reaction was initiated by theaddition of Mg+2 and then quenched and measured as in theoptimum temperature step. One unit of DNA-dependent

DNA polymerase activity is defined as the incorporation of10 nmol of dNTPs into acid-insoluble product in 30 min.

35exonuclease activity 35 exonuclease activity ofGca pol I was assayed using two substrates. One of thesubstrates was a 33-mer single-strand DNA (ssDNA) andthe other was 32/14 double-stranded DNA (dsDNA)containing a single terminal mismatch at 3 end (Table 1).In these assays, KF and KF exo were used as positive andnegative controls respectively. Exonuclease assays werecarried out in 6-l reaction volume containing polymeraseactivity buffer, 12 nM DNA substrate, 100 nM enzyme, and6 mM MgCl2for 1 and 5 min. The reactions were quenchedand visualized as above.

5nuclease activity The 5 nuclease activity of Gca pol Iwas measured on the substrates with a flap at the 5 end, the

preferred substrate for cleavage by the 5nuclease (Table1)using E. coli pol I as a positive control. Reaction mixture(6 l) contained either one of the DNA substrates (10 nM),

polymerase activity buffer, and 6 mM MgCl2, and thereactions were initiated by the addition of 2 l of each

Appl Microbiol Biotechnol (2009) 84:105117 107


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Table 1 The DNA substrates used in this study for enzymatic characterization

Template-primer 33p16/16p-mer used for the polymerase activity assessment by TCA precipitation assay

Template-primer 32/14-mer used for the polymerase activity assessment by primer extension assay

Template-mismatch primer 32/14-mer used for the 3 5 exonuclease activity assessment

Single Strand DNA 33-mer used for the 3 5 exonuclease activity assessment

Template (RNA)-primer (DNA) 32/14-mer used for the RNA-dependent DNA polymerase activity

assessment by primer extension assay

Template-primer-blocker (52/14/42-mer) containing four nucleotide gap and flap requiring next incoming

correct nucleotide as dATP and the numbering scheme of template-nucleotide

Template-primer-blocker (28/14/18-mer) containing 4 nucleotide flap

Template-primer-blocker (32/14/18-mer) containing 4 nucleotide gap and flap

Template-primer-blocker (32/14/14-mer) containing 4 nucleotide gap

Asterisk indicates both the labeled strand and the labeled position



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enzyme (300 nM) at 60C for Gca pol I and 37C for E.colipol I, respectively, for various times from 5 s to 10 minwith 28/14/18-mer DNA and 2 min with 52/14/42-merDNA. The reactions were quenched and visualized asabove.

RNA-dependent DNA polymerase activity This activity of

Gca pol I was determined using either 6 mM MgCl2 or7 mM MnCl2, using HIV-RT as positive and KF as negativecontrols. The reaction mixture (6 l) contained polymeraseactivity buffer, 50 M of each dNTP, and 5 nM 32/14-merheteropolymeric RNA/DNA (Table 1). The reactions wereinitiated by the addition of 150 nM of each enzyme andincubated at 37C (KF) and 60C (Gca pol I) for 1 and5 min (0.5 and 1 min for HIV-RT). The reactions werequenched and visualized as above.

Fidelity of DNA synthesis The error proneness (fidelity) ofGca pol I was determined using primer extension assay

both in the presence of either three or four dNTPsessentially as described by Preston et al. (1988) using Mtb

pol and KF controls and in the presence of one dNTPseparately. A reaction mixture (6 l) contained polymeraseactivity buffer, 6 mM MgCl2, 50 nM enzyme, either all fourdNTPs (50 M each) or only three dNTPs (dATP, dGTP,and dTTP) or only one dNTP (dATP or dTTP or dCTP ordGTP) at 50 M each, and 250 nM of 32/14-mer DNA.The reactions were initiated by the addition of enzyme andincubated at 37C for all enzymes and at 60C just forGca

pol I. Two time points (30 s and 2 min) were measured forall enzymes, following which the reactions were quenched

and visualized as above.

Determination of KDDNA To determine the binding affinity

of template primer to theGca pol I, gel mobility shift assaywas performed. The binding of 32/14 template primer tovarious amounts of the enzyme from 3.125 to 100 ng wascarried out in the polymerase activity buffer using 50100 pM of DNA. Products were fractioned, visualized, andevaluated (Gangurde and Modak 2002). Percent bindingvalues were then used for the determination of KD

DNA byinterpolation, using nonlinear regression for one-site bind-ing (hyperbola) with the GraphPad Prism software.

Determination of KDdNTP and KpolPresteady-state kinetic

parameters KDdATP (dissociation constant for dATP) and

Kpol(first-order rate constant) were determined as describedby Potapova et al. (2006). Single-turnover measurements ofnucleotide incorporation by Gca pol I was carried out atroom temperature (25C), using a rapid quench-flowinstrument (KinTek Corp.). Seven different dATP concen-trations ranging from 2 to 100M were used forKD(dATP)determination. Reaction mixture (56.7 l) contained poly-

merase activity buffer, 6 mM MgCl2, 100 nM 32/14-merDNA (Table1), and 50 nM Gca pol I. The reactions wereinitiated by mixing different concentrations of dATP andincubated from 5 ms to 2 min. The reactions were quenchedusing 15 mM EDTA at the desired time intervals andvisualized as above. The data were processed as described

by Astatke et al. (1998).

Dideoxynucleotide sensitivity Primer extension assays wereperformed to determine ddNTP sensitivity using 32/14-merDNA in the presence of either of all four dNTPs at 50 Meach and 700 M of one of the four ddNTPs. The reactionswere carried out in a final volume of 6 l and contained

polymerase activity buffer, 6 mM MgCl2, 6 nM DNA, and10 nM enzyme. The reactions were initiated by the additionof enzyme and incubated for 5 min at 60C. Assays weredone on both Gca pol I and Gca pol I-F712Y mutantenzyme. Four different ddNTP concentrations (6, 12, 20,and 40 M) were used for F712Y mutant protein. Reac-

tions were quenched and visualized as above.

Strand displacement activityThe strand displacement DNAsynthesis byGcapol I and Gca pol I-Y721F mutant enzymewas assayed by incubating 100-nM 32/14/14-mer gappedDNA and 32/14/18-mer flap structured DNA (Table 1) with50 nM of enzyme in a buffer containing polymerase activity

buffer, 6 mM MgCl2at 60C for 30 s and 2 min. E. colipol IandMtb pol I were used as positive and negative controls at37C, respectively. The DNA synthesis was initiated by theaddition of four dNTPs (50 M each, final concentration).The reactions were quenched and visualized as above.

Results

Nucleoide sequence, expression, and purification TheDNA polymerase I gene of Geobacillus caldoxylolyticusTK4 was cloned and biochemically characterized. Nucleo-tide sequence analysis revealed an ORF of 2,637 bp (56.6%GC) including the stop codon, which encodes a polypeptideof 878 amino acids, corresponding to a molecular mass of99,600 Da. The gene sequence has been submitted to theGenBank database under the accession number DQ340803.The amino acid sequence shared about 90% similarity toDNA polymerases from Bacillus species. The similarity ofGcapol I sequence to Taq, E. coli, and Mtb polymerases is59%, 57% and 59%, respectively. However, several gaps inthe 35 exonuclease domain were noted. All theexonuclease motifs existing in E. coli polymerase I areeither absent or altered in Gca pol I likeBstpol I (Aliotta etal.1996). Expression ofGcapol I, its mutant forms (F712Yand Y721F), and Mtb pol I was achieved in E. coli BL21



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(DE3)pLysS.E. coli pol I, KF, and KF exo were purifiedfrom E. coli JM378 according to Joyce and Derbyshire(1995). All these enzymes were purified to near homoge-neity, as judged by SDS-PAGE.

General properties of Gca pol I Gca pol I exhibits amaximum activity at 60C (Fig.1a). The residual polymer-

ase activity was measured after incubation ofGca pol I for5 min at indicated temperatures (Fig. 1b). The enzymeremained stable up to 6065C. However, it rapidly lostactivity at temperatures above 70C. This temperature isabove the optimal growing temperature (60C) ofGca TK4and it shows that Gca pol I has a optimum DNA

polymerase activity around the growing temperature ofGcaTK4. A total loss of activity was observed above 75C.

Specific enzyme activity forGca pol I was found to be495,000 U/mg protein with Mg+2. The enzyme displays alower polymerase activity with Mn2+. The pH optimum forthe enzyme activity was found to be in the range of 7.68.8.

The other thermostable DNA polymerases studied so farhave also been reported to have an optimal activity ataround alkaline pH values, generally in the range of 8.09.0(Uemori et al. 1993).

35 exonuclease activity 35 exonuclease activity ofGcapol I was investigated measuring the rate of excision ofa single mispaired base from 3end of mismatched dsDNAas well as the cleavage rate of ssDNA. KF and KF exo

enzymes served as positive and negative controls for thisactivity, respectively. The KF was able to efficientlydegrade the 33-mer oligonucleotide into products less than

10 nt in length within the time of the reaction (Fig.2a), andit also removed the mismatched nucleotide from thedsDNA substrate. On the other hand, Gca TK4 pol I andthe KF exo were not able to degrade these substrates evenafter extended period of incubation (Fig. 2a, b).

5nuclease activity The 5 nuclease activity of Gca pol Iwas assayed using both 3- and 5-labeled DNA substrates.Gca pol I cleaved the 28/14/18-mer DNA substrate at two

positions at the 5 end of the flap while E. coli pol Ipredominantly cleaved between the first two paired bases,giving rise to a 5-bp cleavage product. However, the cleavage

was found to occur equally between the first two paired basesand also just before the paired bases with Gca pol I. Thisresults in a mixture of 4 and 5 bp cleavage products (Fig.3a).The nick-flap DNA was cleaved much more rapidly by Gca

Fig. 1 Effect of temperature onGcapol I activity (a) and deter-mination of thermal stability (b).Thermal stability ofGca pol Iwas measured with enzymesamples incubated for 5 min atvarious temperatures from 55 to85C with 5C increments andsubsequently chilled on ice. Theresidual enzyme activity of theseheat-treated enzymes was mea-sured by primer extension. Lane

Pshows the position of 14-merprimer and lane Cshows theproduct of primer extension as-say from 14-mer to 32-mer withuntreated Gca pol I as a control



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pol I than theE. coli pol I. Each enzyme cuts the overhangpart of the flap strand on 56/14/42-mer DNA and thencleaves nucleotides in the direction of 53 one base at atime (Fig.3b). TheGcapol I andE. colipol showed a similarcleavage activity by blocker DNA but not by flap DNA.

RNA-dependent DNA polymerase activity RNA-dependentDNA polymerase activity ofGca pol I was measured in the

presence of either Mg2+ or Mn2+ as cofactor. HIV-RT andE. coli pol I were used as controls. While HIV-RTcompleted the synthesis in 30 s, Gca pol I and E. coli pol

Fig. 2 Measurement of 35exonuclease activity. 35exonuclease activity ofGca polI was measured with both 33-mer ssDNA (a) and 32/14-merterminal-mismatch containingDNA substrate (b) at 60C for 1and 5 min. DNA sequences ofthe DNA substrates are shown

under the figure. KF and KFexo were also analyzed ascontrols at the same reactioncondition at 37C. Lane Mshows the position of 33 and18-mer ssDNA

Fig. 3 Measurement of 5

nuclease activity. 5nucleaseactivity ofGca pol I was mea-sured with 5-flap containingDNA substrates shown in a andb. a Activity was measuredusing a 28/14/18-mer DNAsubstrate from 5 s to 10 min,where the 5end flap containingstrand (18-mer) was labeledwith 32P at 5end. b Activitywas measured using a 56/14/42-mer DNA substrate for 2 min.DNA sequences of the substrateare shown belowa and on the

right ofb. Lane Mshows thepositions of 18-mer ssDNA(panel a) and 42-mer ssDNA(panel b). Line E and G (panel

b) show E. colipol I and Gcapol I respectively



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I added two nucleotides efficiently into RNA template withMg+2 (Fig.4a). However, both Gca pol I and E. colipol Ishowed a higher RNA-dependent DNA polymerase activitywith Mn2+ (Fig. 4b), and both enzymes completed DNAsynthesis efficiently.

Fidelity of Gca pol IReactions to test enzyme fidelity werecarried out with individual dNTP separately at two time

points and at two different temperatures. The first available

base to the incoming nucleotide on the template wasthymidine (T), and thus, dATP as the next incoming dNTPwould give rise to a 15-mer product. In the presence of theother three dNTPs (dTTP, dGTP, and dGTP), no primerextension is expected. It can be seen from Fig.5a thatGca

pol I shows maximum extension with dATP as substrate.Results shown in Fig. 5 also show that the fidelity ofenzyme was changed depending on the temperature. Gca

pol I was found to make more mistakes at 60C than at 37C.From the results of this assay, we calculated the rate ofmismatch incorporation ofGca pol I to be one nucleotide

per 40,000 nucleotides added. We obtained a similar ratewith the other three dNTPs (dATP, dGTP, and dTTP) usingKF andMtb pol I as controls. From the results of this assay,we found that the error rate ofGca pol I is comparable tothat of KF and slightly more than Mtbpol I.

Template primer binding affinity and presteady-statekinetics The classical gel retardation assay was used todetermine the DNA binding affinity of the Gca pol I. TheenzymeDNA dissociation constant KD

DNA was deter-mined by plotting percent template/primer bound as afunction of enzyme concentration and then fitting thedata to a single site association hyperbola. Retardation ofthe labeled template/primer band indicates the enzyme

Fig. 4 Determination of RNA-dependent DNA polymerase ac-tivity. RNA-dependent DNA

polymerase activity ofGca pol Iwas measured by primer exten-sion assay with 32/14-mer het-eropolymeric RNA/DNAsubstrate. HIV-RT and KF wereused as controls in the same

reaction conditions at 37C.Lane Pshows the position of32-mer generated by primer ex-tension assay by DNA/DNAsubstrate as a control and lane Cshows the 14-mer primer

position

Fig. 5 Measurement of fidelity of DNA synthesis. Fidelity ofGcapolI was determined with 32/14-mer DNA substrate. Single nucleotideincorporation by the Gca pol I was studied by supplying 50 M ofeach of the four dNTPs in individual reactions (a). Incorporation ofthree nucleotides by Gca pol I, KF, and Mtb pol I was studied (b) by

supplying 50M dATP, dCTP, and dTTP but no dGTP with the same32/14-mer DNA substrate. All reactions were performed at 37C and60C forGcapol I and 37C for KF and Mtbpol I for 30 s and 2 min.The relevant template sequence is shown at the right side of the gel

picture;lane Pshows the position of the primer



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DNA complex. The KDDNA values for Gca pol I was

determined to be 0.192 nM. The KDdNTP and Kpol values

of Gca pol I were determined by using single-turnoverkinetic for incorporation of dATP. The KD

dNTP and Kpolvalues were measured to be 22.64 M and 24.99 s1,respectively.

ddNTP sensitivity To determine the ddNTP sensitivity forGca pol I, we used 700-M ddNTP concentration ofdesired ddNTP and 50-M dNTP concentration to providea ratio of 14:1, respectively. As seen from the results, nativeGca pol I is not sensitive to ddNTPs (Fig. 6a). Gca pol Icontains a phenylalanine residue at the position equivalentto Phe762 ofE. colipol I. We investigated if the mutation ofPhe712 in Gca pol I to tyrosine would alter the dideo-xynucleotide discrimination. This property was examined

by monitoring synthesis on the 32/14-mer DNA in thepresence of all four dNTPs (120 M) and individualddNTP (from 6 to 40 M). As seen in Fig. 6b, at each

ddNTP concentration, Gca pol I-F712Y mutant enzymerecognizes the ddNTPs effectively and discriminatesagainst it as seen from the arrest of further DNA synthesis.Our data show that the preference for the dNTP overddNTP forGca pol I is lost due to the F712Y mutation. In

fact, in some cases although the concentration of ddNTPswas 20 times less than dNTP, F712Y incorporates ddNTPsefficiently.

Strand displacement activity The strand displacement syn-thesis activity ofGca pol I and its Y721F mutant form wasdetermined using four-nucleotide-gap containing blocker

and four-nucleotide-flap DNA substrates. The results inFig. 7 show that Gca pol I and its mutant form initially

pause after filling the gap (marked as 20-mer) likeMtbpol I(marked as 18-mer). Both Gca pol I and its mutant form areable to complete the displacement of the blocking strandand synthesize a full-length product. It is clear that there isa difference between Gca pol I and its mutant form in theirability to catalyze the strand displacement synthesis, andthe rate of displacement by the Y721F mutant enzyme washigher compared to Gca pol I. The presence of a flapsequence at the 5 end of the blocker does not affect thestrand displacement DNA synthesis activity of Gca pol I

and its mutant form. These results suggest that Phe721 isrequired only for the initiaton of the strand separation orcontinuously during the process of strand displacementsynthesis in Bacillus. E. coli pol showed high stranddisplacement activity butMtb pol I did not in all conditions.

Fig. 6 Measurement of sensitivity to ddNTPs. ddNTPs sensitivity ofGcapol I was measured by primer extension assay. a Reaction withGcapol I using 6 nM 32/14-mer DNA substrate and 50 M each ofthe four dNTPs and 700 M of each ddNTP in separate reactions(labeled on top of each lane). b Reaction with Gcapol I-F712Y using

120 M of all four dNTPs and individual ddNTPs in separate reactions atconcentrations ranging from 6 to 40M. All reactions were performed at60C for 5 min. Lane P shows polymerase reaction as a control (withonly dNTPs, without the addition of ddNTPs). The complementersequence of the 32-mer template strand is shown on the right



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Discussion

The complete gene sequence of the polA gene from G.caldoxylosilyticus TK4 was determined, and typical motifsand highly conserved catalytic residues are observed in the5 nuclease and 53 polymerase domains. DNA-dependent DNA polymerase activity assay showed thatGca polymerase I was quite efficient and catalyzed theincorporation of about 25 nt/s. High specific activity withMg2+ indicates that it is preferred as a cofactor. The valuesfor KD

DNA, KDdNTP, and Kpolare similar to those reported

for Bacillus and other DNA polymerases Is (Arrigo et al.2002; James et al.1996). Thermal stability studies showedthat Gca pol I exhibited maximum activity close to theoptimal growth temperature (60C) of the Gca TK4. Thus,Gca pol I can be expected to have optimal activity underthe natural hot spring environment in which the organismgrows. The same has also been observed before for pol Ienzymes from other Bacillus species (Uemori et al 1993;Phang et al. 1995). The same held true for pH optimum(8.0) as for all known pol I enzymes.

Significant sequence similarity was found between GcaTK4 and Bacillus DNA polymerase Is compared to the

other polymerases. The deduced amino acid sequence ofGca

pol I was compared to amino acid sequences from other polIs fromE. coli,Mtb,Taq,Bst, andBca, and the similarity was

between 57% (E. coli) and 89% (Bst), respectively (Table2).All conserved amino acids for the 35 exonucleaseactivity in E. coli pol I are either absent (equivalent toD355 and E357 in E. coli) or altered (equivalent to D424 andD503 in E. coli) inGcapol I (Derbyshire et al.1991). Theseresidues are also absent or substituted in the known pol Is of

Bacillus species. These residues are required for 35exonuclease activity, and mutation of any one of theseresidues in KF abolishes 35 exonuclease activity. These

properties of Gca pol I were urther confirmed by 3 5exonuclease activity assays on mismatch-containing dsDNAand ssDNA substrates forGca pol I in this study and for the

pol I enzymes from other Bacillus species in earlier studies(Uemori et al. 1993; Aliotta et al. 1996). Proteins with anonfunctional domain can probably tolerate more mutationsin that domain, and the 35 exonuclease domain in

Bacillus DNA pol I has more substitutions than the 5nuclease and polymerase domains when compared to KF.

The 53 exonuclease is known to be a structure-specific nuclease that cleaves a 5-displaced strand at the

Fig. 7 Measurement of strand displacement activity. Strand displace-ment activity ofGca pol I and its Y721F mutant form was measuredwith flap structured (a) and gapped DNA (b) for 30 s (column 1) and2 min (column 2) for each individual enzyme. DNA sequences of theDNA substrates are shown on the left and right of the figure. Lane Mis a marker for the positions of 14-mer and 18-mer; lane Cshows

polymerase activity ofGca pol I using 32/14-mer DNA substrate for1 min. Sections A and B show the strand displacement DNA synthesis

byGcapol I, its mutant form, E. colipol I, and Mtbpol I, respectively.

The product of the four-nucleotide gap-filling reaction was mostly 18-and 20-mer position on both flap and blocker DNA forGca pol I andits mutant form, respectively. The product of primer extension activitywith these two substrate was mostly 18-mer for Mtb pol I. In allconditions, E. coli pol I completed the synthesis within the reactiontime. The mutant enzyme completed the gap filling and stranddisplacement DNA synthesis faster than wild-type Gca pol I on boththe blocker and flap DNA



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junction between single-stranded and duplex regions. BothGca pol I and E coli pol I cleaved the 18-mer between thefirst two paired bases from the 5 end to yield a labeled 5-mer, but Gca pol I produced the same amount of 4-mer

product. We have found that Gca pol I cuts both betweenthe two paired bases nearest to the 5 end and just before

paired bases equally on 28/14/18-mer flap DNA. We foundthat theGca pol I degrades the DNA in direction on 53one base at a time with 56/14/42-flap DNA substrate, justlike E. coli pol I. The 53 exonuclease reaction has astrict requirement for a free 5end on the displaced strand.

However, the upstream template and primer strands aredispensable. There are ten conserved carboxylate residuesin 5 nuclease domain among bacteria and bacteriophage

polymerases, and nine of them are important for nucleaseactivity (Table2; Yang et al. 2001). All these residues areconserved among polymerase Is of Gca TK4 and Bacillusspecies. A primary amino acid sequence comparison of

polymerase I family enzymes has shown a high degree ofconservation of two residues: Ser769 and Phe771 (numberingaccording to E. coli pol I). These residues are found to beessential for strand displacement synthesis (Singh et al.2007). However, in the enzymes that lack strand displace-ment activity, the equivalent positions are occupied bynonhomologous residues. In T7 DNA polymerase, which isdeficient in strand displacement activity (Stano et al.2005),the equivalent positions of Ser769, Phe771, and Arg841 areoccupied by glycine, glutamic acid, and histidine, respec-tively (Singh et al. 2007). The residues Ser719, Tyr721, andArg791 of Gca pol I are equivalent to Ser769, Phe771, andArg841, respectively, in E. colipol I. Thus, only one residue(Y721) is different in Gca pol I than in E. colipol I. Whenwe changed this tyrosine residue to phenyalanine in Gca

pol I, we found that the mutant form showed a greaterstrand displacement activity thanGcapol I, but its activityis lower thanE. colipol I. This may indicate that the rate ofstrand displacement activity of Gca pol I is inherentlyslower than the E. coli enzyme.

There is a variation among family A DNA polymerasesin the RNA-dependent DNA polymerase activity in spite ofsignificant sequence similarity (Myers and Gelfand 1991).These enzymes differ in metal ion requirement and showmore activity with Mn2+. Gca pol I also shows the samecharacteristic for RNA-dependent DNA polymerase activ-

ity. While Gca pol I adds two dNTPs to a RNA/DNAsubstrate efficiently just like KF, all three enzymescompleted the synthesis with Mn2+. However, some

polymerase I enzymes from Bacillus species (Bacilluscaldolyticus EA1 and Caldibacillus cellovorans) wereobserved to show a high activity with RNA/DNA substratewith Mn2+ (Harini et al. 2004). Among other bacteria, TthDNA polymerase (Myers and Gelfand1991) andTne DNA

polymerase (Yang et al. 2002) are known to copy RNA inthe presence of Mn2+. Thus, this property varies amongfamily A DNA polymerases. Error rates during DNAsynthesis depend upon the family to which the DNA

polymerase belongs. The error rates in PCR reaction for KF(Scharf et al.1986) andT. aquaticusDNA polymerase werereported to be 8105 and 2104 (Saiki et al. 1988),respectively. We calculated the value of error rate forGca

pol I to be 2.5105, which was between E. colipol I andTaq pol I. This indicates that Gca pol I, although lacking

proof reading activity, may be a high fidelity enzyme.Mutation of Phe712 to Tyr712 in Gca pol I produces an

enzyme capable of incorporating dideoxynucleotides, whilewild-type Gca pol I strongly discriminates against ddNTPs.

Table 2 Similarity of conserved amino acid motifs involved in the 35exonuclease and 53polymerase activity in various bacterial DNApolymerases

Species Similarity toGcaTK4 pol I(aa, %)

35conservedresidues

Residue equivalentto E. coliPhe762

Residuesequivalent to

E. coliPhe771

(in bold)

References

G. caldoxylosilyticusTK4 - - AK Phe (F712) SY721R This study

B. stearothermophilus 89 VE - I Phe (F710) SY719R Aliotta et al. (1996)

B. stearothermophilus 89 VEAK Phe (F710) SY719R James et al. (1996)

B. caldotenax 89 - - AK Phe (F711) SY720R Uemori et al. (1993)

Geobacillus kaustophilus 89 - - AK Phe (F712) SY721R GenBank; YP148583

Geobacillussp. MKK 90 - - AK Phe (F710) SY719R Khalaj-Kondori et al. (2007)

Oceanobacillus iheyensis 80 - - TK Phe (F714) SY723R GenBank; NP693084

Thermus aquaticus 59 - - L E Phe (F667) SH676R Lawyer et al. (1989)

Mycobacterium tuberculosis 59 DRAR Tyr (Y737) SY746R Polesky et al. (1990)

E. coli 57 DEDD Phe (F762) S769F771R841 Singh et al. (2007)

Rhodothermus marinus 56 DEDD Tyr (Y757) SW766R Thorarinn et al. (2001)



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Mtb pol I, which has tyrosine residue at the positionequivalent to Phe762 of E. coli pol I, has been shown toincorporate dideoxynucleotides more rapidly than E. coli

pol I and with approximately the same frequency as that ofT7 DNA polymerase (Mizrahi and Huberts1996). Gca polI was found not to be sensitive to ddNTPs and thesubstitution of the tyrosine 712 to phenylalanine was found

to confer sensitivity to ddNTPs. Its mutant form is able todistinguish the ddNTPs from dNTPs much better than Gca

pol I. This makes it an attractive enzyme for use in DNAsequencing. Mutations that increase the ability of a DNA

polymerase I of Bacillus species to incorporate ddNTPsinto DNA have not been previously described. In earlierstudies (Tabor and Richardson1995; Li et al. 1999), it wasdetermined that the critical phenylalanine/tyrosine residuedefining the ability of Pol I-type DNA polymerases todistinguish between deoxy- and dideoxyribonucleotides islocated on the O-helix facing into the crevice responsiblefor binding dNTPs and DNA. Amino acid residues

equivalent to Gca pol IPhe712 in Bacillus species andsome polymerase Is from other bacteria are listed in Table2.DNA polymerase Is from different Bacillus species have

phenylalanine at this position on the O-helix. Bacteria withphenylalanine at this position grow more rapidly than thosehaving tyrosine at the same position such as M. tuberculosisand Mycobacterium leprae (Tabor and Richardson1995).

In conclusion, Gca pol I exhibited kinetic propertiescomparable to E. coli pol I, and the residue F712 of Gca

pol I (equivalent to F762 in E. coli pol I) was found to beresponsible for ddNTP sensitivity. WhileGcapol I does notshow any detectable 35 exonuclease activity, its 5

nuclease activity was similar to E. coli pol I when theblocker DNA was used as a substrate. Although the stranddispacement synthesis activity of native Gca pol I waslower than that ofE. colipol I, higher activity was seen bymutating the Tyr721 to Phe in Gca pol I. To the best of ourknowledge, mutational studies involving these two resi-dues, viz. F712 (ddNTP sensitivity) and Y721 (stranddisplacement synthesis), have not been previously reportedin any DNA pol I from Bacillusspecies.

Acknowledgments We thank Dr. M. Claeyensens and Dr. K.Rumsfold for technical assistance. This study was supported by grants

from The Scientific and Technical Research Council of Turkey(TUBTAK-105T216) and Karadeniz Technical University (BAP-2005.111.004.1) and a scholarship to C. Sandalli from TUBTAK.

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