Amino Acid Residues Critical for the Interaction Between Bacteriophage T7 DNA Polymerase and Escherichia Coli Thioredoxin

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Amino Acid Residues Critical for the Interaction between Bacteriophage T7 DNA Polymerase and Escherichia coli Thioredoxin

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Citation Himawan, Jeff S., and Charles C. Richardson. 1996. “Amino Acid Residues Critical for the Interaction between Bacteriophage T7 DNA Polymerase andEscherichia coliThioredoxin.” Journal of Biological Chemistry 271 (33): 19999–8. https://doi.org/10.1074/ jbc.271.33.19999.

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41483387

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 33, Issue of August 16, pp. 19999–20008, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Amino Acid Residues Critical for the Interaction between Bacteriophage T7 DNA Polymerase and Escherichia coli Thioredoxin*

(Received for publication, March 28, 1996)

Jeff S. Himawan‡ and Charles C. Richardson§ From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Upon infection of Escherichia coli, bacteriophage T7 nificantly by mutation, then complex formation with the second annexes a host protein, thioredoxin, to serve as a pro- protein would be destroyed. Theoretically, a productive com- cessivity factor for its DNA polymerase, T7 gene 5 pro- plex could be formed once again by an alteration in the second tein. In a previous communication (Himawan, J., and protein that structurally compensates for the original muta- Richardson, C. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, tion. Therefore, by mutating one protein of a complex and 9774–9778), we reported that an E. coli strain encoding a selecting for extragenic suppressor mutations in the second Gly-74 to Asp-74 (G74D) thioredoxin mutation could not protein, one should be able to identify the contact points be- support wild-type T7 growth and that in vivo, six muta- tween the two proteins. tions in T7 gene 5 could individually suppress this G74D We (1) have used extragenic suppressor analysis to investi- Downloaded from thioredoxin defect. In the present study, we report the gate the interactions between two proteins that are involved in purification and biochemical characterization of the DNA replication in Escherichia coli infected with bacterio- G74D thioredoxin mutant and two suppressor gene 5 phage T7. Similar to our studies, other workers have also used proteins, a Glu-319 to Lys-319 (E319K) mutant of gene 5 suppressor analysis to study protein-protein interactions in the protein and an Ala-45 to Thr-45 (A45T) mutant. The sup- E. coli DNA replication system (2) and also in the DNA repli- pressor E319K mutation, positioned within the DNA http://www.jbc.org/ polymerization domain of gene 5 protein, appears to cation system of the yeast Saccharomyces cerevisiae (3). Spe- suppress the parental thioredoxin mutation by compen- cifically, we have been investigating by suppressor analysis the sating for the binding defect that was caused by the interaction between T7 gene 5 protein and E. coli thioredoxin. G74D alteration. We suggest that the Glu-319 residue of T7 gene 5 protein, the DNA polymerase of phage T7 (4, 5), has T7 gene 5 protein and the Gly-74 residue of E. coli thi- two enzymatic activities: a nonprocessive 5Ј to 3Ј DNA polym- oredoxin define a contact point or site of interaction erase activity (6–8) and a 3Ј to 5Ј exonuclease activity (7, 9). between the two proteins. In contrast, the A45T muta- During infection of E. coli, T7 annexes the host protein thiore- by guest on October 5, 2019 tion in gene 5 protein, located within the 3؅ to 5؅ exonu- doxin, a general protein disulfide oxidoreductase (10), as a clease domain, does not suppress the G74D thioredoxin processivity factor for polymerization of nucleotides (8, 11–14). mutation by simple restoration of binding affinity. Thioredoxin also greatly stimulates the 3Ј to 5Ј exonuclease Based upon our understanding of the mechanisms of activity of T7 gene 5 protein on double-stranded DNA (7, 9). suppression, we propose a model for the T7 gene 5 pro- We have focused our studies on the interactions between T7 tein-E. coli thioredoxin interaction. gene 5 protein and thioredoxin in order to understand how thioredoxin confers processivity upon gene 5 protein. Unlike other more complex DNA replication systems, such as those of The concept of using genetic or suppressor analysis to inves- bacteriophage T4, E. coli, and eukaryotes (15–18), the relative tigate protein-protein interaction can be described as follows. If simplicity and experimental tractability of the phage T7 DNA two proteins form a complex, then there must exist a contact replication system affords the attractive possibility of under- point, or more likely, several contact points between them. standing processivity comprehensively at a molecular level. In These contact points would be defined by certain amino acid this work, we have complemented our previous genetic analysis residues of one protein that must be physically adjacent to of the interaction between T7 gene 5 protein and E. coli thiore- certain amino acid residues of the other protein. If a contact doxin (1) with a biochemical analysis of the same interaction. point amino acid from one protein is structurally altered sig- We began our previous investigation by using E. coli thioredoxin mutants that were unable to support wild-type (WT)1 T7 * This work was supported in part by National Institutes of Health growth to select for suppressor strains of phage T7 that con- Grant AI-06045 and Department of Energy Grant DE-GF02- tained a compensating mutation in gene 5. We found that an E. 88ER60688. The costs of publication of this article were defrayed in part coli strain containing a glycine 74 to aspartate 74 substitution by the payment of page charges. This article must therefore be hereby (G74D) in thioredoxin could not support the growth of WT T7 marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. phage. Furthermore, we found that six different mutations in This work is dedicated to the memory of Lewis Thomas, whose books T7 gene 5 could individually suppress the G74D alteration in greatly inspired one of us (J. S. H.). thioredoxin. Three of the six suppressor mutations (E319K, ‡ Supported, in part, by a John Stauffer Graduate Fellowship from E319V, Y409C) were positioned within the putative DNA po- the Stauffer Charitable Trust. § Consultant to Amersham Life Science Inc., which has licenses from lymerization domain of T7 gene 5 protein, and the other three Harvard University to commercialize DNA polymerases for use in DNA suppressor mutations (A45T, V3I, V32A) were positioned sequencing. To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1864; Fax: 1 The abbreviations used are: WT, wild type; PCR, polymerase chain 617-432-3362; E-mail: [email protected]. reaction. 19999 20000 T7 DNA Polymerase-E. coli Thioredoxin Interaction within the 3Ј to 5Ј exonuclease domain. In this paper, we have The amount of protein in a given solution was determined by the attempted to explain, at a biochemical level, the mechanism of method of Bradford (31), using the Bio-Rad protein assay kit and using suppression for two representative suppressor mutations bovine plasma ␥ globulin (Bio-Rad) as a standard protein. An ice-cold solution of 10 mM Tris-Cl, pH 7.5, 5 mM dithiothreitol, and 0.5 mg/ml (E319K and A45T). bovine serum albumin was used to dilute enzymes immediately prior to starting the desired reactions. Polyacrylamide gel electrophoresis fol- EXPERIMENTAL PROCEDURES lowed by Coomassie staining was performed by standard techniques Bacterial Strains, Bacteriophage Strains, and Plasmids—E. coli (28), and polyacrylamide gel electrophoresis followed by silver-staining JH20 (⌬trxA307,pcnB80), E. coli HMS231 (trxAϩ,pcnBϩ), E. coli was performed using the PhastSystem from Pharmacia. SB2111 (a strain harboring the plasmid pBR325trxA11), E. coli Expression and Purification of the Gly-74 to Asp-74 (G74D) Thiore- MV1190 (FЈ,supE), WT bacteriophage T7, phage T7trx5 (a T7 phage doxin—E. coli strain AN1, harboring the plasmid pBR325trxA11, was that has a WT thioredoxin gene inserted in its genome), phage T7–5- used to overproduce the G74D thioredoxin. By slightly modifying pre-

E319K, phage T7–5-A45T, bacteriophage P1vir, and plasmid vious protocols that were used to purify WT thioredoxin (11, 19, 32, 33), pBR325trxA11 (a plasmid encoding a Gly-74 to Asp-74 thioredoxin we developed a six-step, three-column procedure to purify the G74D mutant) were described previously (1). E. coli A307, ompT, a derivative mutant thioredoxin. Throughout this purification procedure, all solu- of E. coli A307 (19) that contains an additional mutation in the ompT tions containing the G74D thioredoxin were kept at 0–4 °C. Two liters gene, bacteriophage M13mGP1–2, a derivative of phage M13mp8 that of E. coli AN1(pBR325trxA11) cells were grown at 37 °C in rich media contains the T7 RNA polymerase gene under the transcriptional control (2% Tryptone, 1% yeast extract, and 0.5% NaCl) with vigorous shaking. of the lac promoter (8), and plasmid pGP5–5, a derivative of plasmid Following overnight growth, the cell culture was centrifuged to collect pACYC177 (20) that contains the WT T7 gene 5 under the transcrip- the cells, and the cells (wet weight of 10 g) were resuspended in ice-cold tional control of two tandem T7 promoters (⌽1.1A and ⌽1.1B), were buffer containing 50 mM Tris-Cl, pH 8.2, 20 mM EDTA, 10% sucrose. obtained from Dr. Stanley Tabor (Harvard Medical School). Plasmids The solution of resuspended cells was frozen by treatment with liquid pGP5–5-E319K and pGP5–5-A45T, both derivatives of pGP5–5, were nitrogen, and the frozen cells were thawed by an overnight incubation constructed as described below. at 0 °C. This freeze-thaw procedure selectively releases thioredoxin ϩ E. coli AN1 (⌬trxA307,pcnB ) was constructed by using phage P1vir from the cells (34). The cell debris was removed by centrifugation to to transduce the ⌬trxA307 allele from JH20 into HMS231. The presence produce fraction I (freeze-thaw supernatant). Residual amounts of DNA Downloaded from of the thioredoxin deletion (⌬trxA307) within AN1 was verified by were removed from fraction I (35 ml) by the addition of streptomycin several methods. First, AN1 was unable to support the growth of WT T7 sulfate (4% final concentration), allowing selective precipitation of the but was able to support the growth of T7trx5, indicating a defect in the DNA by centrifugation to generate fraction II (streptomycin sulfate thioredoxin gene. Second, amplification by polymerase chain reaction supernatant). Ammonium sulfate was added to fraction II (3.5 g/35 ml) (PCR) of the chromosomal DNA surrounding the thioredoxin gene to precipitate proteins, generating fraction III (ammonium sulfate su- showed the presence of a deletion of the appropriate size. Third, restric- pernatant). Fraction III (35 ml) was dialyzed against 2 liters of 10 mM

tion enzyme analysis of the PCR-generated DNA fragment confirmed Tris-Cl, pH 8.0, 1 mM EDTA, 1 mM ␤-mercaptoethanol (buffer A) and http://www.jbc.org/ that the deletion was positioned correctly. was applied onto a DEAE-cellulose column (4.9 cm2 ϫ 8 cm) that was E. coli AN1(pBR325trxA11), the strain used to purify the Gly-74 to equilibrated with buffer A. Bound G74D thioredoxin was eluted by a Asp-74 thioredoxin (G74DTrxA), was constructed by purifying the plas- 250-ml continuous gradient of NaCl (25 mM to 500 mM) in buffer A. The mid pBR325trxA11 from E. coli SB2111 and by transforming fractions containing thioredoxin were pooled (70 ml), concentrated by pBR325trxA11 into E. coli AN1. Strain SB2111 was not used for the ammonium sulfate precipitation (33 g/70 ml), and dissolved in 1 ml of 10 production of the G74DTrxA, because the pcnBϪ mutation within mM Tris-Cl, pH 7.5, 0.1 mM EDTA, 1 mM ␤-mercaptoethanol (buffer B)

SB2111 reduces the intracellular copy number of plasmid to generate fraction IV. Fraction IV (1 ml) was subjected to gel filtration by guest on October 5, 2019 pBR325trxA11 (1). Consequently, SB2111 produces less G74DTrxA through a Sephadex G-50 column (1.8 cm2 ϫ 50 cm) that was equili- than AN1(pBR325trxA11). brated in buffer B, and fractions that contained thioredoxin were pooled Materials—AmpliTaq DNA Polymerase, purchased from Perkin- (5 ml) to generate fraction V. Using the fast protein liquid chromatog- Elmer, was used for in vitro DNA amplification by PCR (21). Unless raphy (FPLC) system from Pharmacia, fraction V (5 ml in buffer B) was specified otherwise, all oligonucleotides used in this work were provided loaded unto a Mono Q HR 5/5 column (1-ml bed volume) that was by Dr. Alex Nussbaum (Harvard Medical School). Nonradioactive nu- equilibrated in buffer B. Thioredoxin bound to the Mono Q matrix and cleotides were purchased from Pharmacia Biotech Inc. [35S]dATP␣S was eluted by a continuous gradient of NaCl (10–300 mM) in buffer B. was obtained from DuPont NEN. [␥-32P]ATP and [3H]dTTP were pur- Under these conditions, thioredoxin (fraction VI) eluted at 85 mM NaCl, chased from Amersham Life Science. The restriction enzymes SapI, and the G74D thioredoxin was greater than 95% pure, as estimated by SnaBI, and HhaI were purchased from New England Biolabs, and denaturing SDS-polyacrylamide gel electrophoresis followed by silver PshAI was purchased from Takara Biochemical Inc. All restriction staining (data not shown). enzyme digests were performed as suggested by the manufacturer. During purification of WT thioredoxin, the presence of the protein Arctic shrimp alkaline phosphatase, T4 DNA ligase, and T4 polynucle- can be detected quantitatively by its ability to reduce certain substrates otide kinase were purchased from U.S. Biochemical Corp. and used (35) or by its ability to stimulate the DNA polymerase activity of T7 under conditions specified by the manufacturer. WT T7 DNA polymer- gene 5 protein (T7 DNA polymerase) (6). In contrast, the G74D mutant ase (WT gene 5 protein or WTg5P) and WT thioredoxin (WTTrxA) were thioredoxin had negligible reduction activity (19) and could stimulate gifts from Dr. Stanley Tabor. Bovine serum albumin was purchased T7 DNA polymerase only at relatively high (10 ␮M) concentrations (36). from Boehringer Mannheim. Isopropyl-␤-D-thiogalactopyranoside was Consequently, during the purification process, the presence of the G74D purchased from U.S. Biochemical Corp. DEAE-cellulose (DE52), phos- thioredoxin was detected by denaturing polyacrylamide gel electro- phocellulose (P11), and DE81 ion exchange paper were purchased from phoresis. A purification table was constructed retrospectively by using Whatman Paper Ltd. Sephadex G-50 and DEAE-Sephadex A-50 were the purified G74D thioredoxin (fraction VI) to estimate the amount of purchased from Pharmacia. mutant thioredoxin that was present in the earlier fractions. In sum- Bacteriophage Methods, DNA Manipulations, and Protein Methods— mary, we estimate that fraction I contained 350 mg of total protein with Transduction by phage P1 was performed as described (22). Bacteri- 1170 units of activity (specific activity of 3 units/mg), and fraction VI ophage T7 was propagated by infecting E. coli cells (23) that were grown contained 3 mg of protein with 180 units of activity (specific activity of in LB media (22) at 37 °C, and phage T7 DNA was prepared as de- 60 units/mg), resulting in approximately a 15% total yield and a 20-fold scribed previously (24). Bacteriophage M13 was propagated as de- purification. One unit of thioredoxin activity was defined arbitrarily as scribed (25). Burst sizes were calculated as described previously the amount of thioredoxin required to stimulate the T7 DNA poly- (26, 27). merase-mediated incorporation of 10 nmol of deoxynucleotides into Agarose and polyacrylamide gel electrophoresis were performed by DNA in a standard DNA polymerase assay containing 5 nM of WT T7 standard techniques (28). The Geneclean kit, purchased from BIO 101, DNA polymerase. Inc. (Vista, CA) was used to purify DNA from fragments of agarose gels Cloning, Expression, and Purification of the Glu-319 to Lys-319 and from other DNA-containing solutions. The Wizard Minipreps DNA (E319K) Gene 5 Protein—DNA from the mutant T7 phage strain T7–5- purification system, purchased from Promega, was used to purify plas- E319K, a T7 phage encoding a glutamate to lysine substitution at mid DNA from cells. Transformation of plasmid DNA into competent E. amino acid residue 319 of its gene 5 protein (1), was purified. The coli cells was performed as described (28). DNA sequencing was per- purified T7–5-E319K DNA was digested with the restriction enzymes formed, using the enzyme Sequenase (U.S. Biochemical Corp.), by the SapI and PshAI, and the resulting 11 DNA fragments were separated Sanger-dideoxy method (29) as described previously (30). by electrophoresis through an agarose gel. The DNA fragment that T7 DNA Polymerase-E. coli Thioredoxin Interaction 20001

TABLE I Purification of the E319K and the A45T mutant gene 5 proteins from E. coli A307, ompT(pGP5–5-E319K) and A307, ompT(pGP5–5-A45T) cells, respectively The amount of protein and the units of DNA polymerase activity were determined as described under “Experimental Procedures.”

Fraction and step Protein Units Specific activity Recovery

mg ϫ103 units/mg % Purification of the E319K gene 5 protein I. Extract 1800 900 500 100 II. Ammonium sulfate 1600 460 290 51 III. DEAE-cellulose 350 360 1030 40 IV. Phosphocellulose 3 50 16,700 6 V. DEAE-Sephadex A-50 1.5 40 26,700 4 Purification of the A45T gene 5 protein I. Extract 600 49 81 100 II. Ammonium sulfate 600 49 81 100 III. DEAE-cellulose 15 20 1347 42 IV. Phosphocellulose 0.1 5.8 58,000 12 contained the E319K mutation (E319K mutant fragment) was purified. mutant T7–5-A45T, a T7 phage harboring an alanine to threonine Similarly, the plasmid pGP5–5 was incubated with the same two re- substitution at amino acid residue 45 of its gene 5 protein (1), was striction enzymes to generate two DNA fragments, and the DNA seg- purified. The T7–5-A45T DNA was digested with the restriction enzyme ment (vector fragment) that did not contain the Glu-319 residue was SnaBI to generate 14 DNA segments, which were separated by agarose purified. This vector fragment was incubated with shrimp alkaline gel electrophoresis. The DNA fragment encoding the A45T mutation phosphatase, and the dephosphorylated vector fragment, unable to was purified (A45T mutant fragment). Plasmid pGP5–5 DNA was also self-ligate, was covalently attached to the E319K mutant fragment by digested with SnaBI to generate two segments, and the DNA fragment Downloaded from incubation with T4 DNA ligase, generating the plasmid pGP5–5- that did not contain the Ala-45 residue (vector fragment) was purified. E319K. The presence of the E319K mutation, a guanine to adenine This vector fragment and the A45T mutant fragment were joined by transition (1) at base number 15,307 of the T7 DNA sequence (37), incubation with T4 DNA ligase, producing plasmid pGP5–5-A45T. within pGP5–5-E319K was verified by DNA sequencing (data not The A45T mutation, a guanine to adenine transition at base number shown). The DNA sequence surrounding the cloning junctions was also 14,485 of the T7 DNA sequence (1), was located within the recognition determined and revealed no other mutations. site (5Ј-GCGC-3Ј) of the restriction enzyme HhaI (the altered base is Plasmid pGP5–5-E319K was transformed into E. coli A307, ompT underlined). This made it possible to verify the presence of the A45T http://www.jbc.org/ and the mutant E319K gene 5 protein (E319Kg5P) was purified from mutation within pGP5–5-A45T by simply detecting the appropriate this strain. E. coli A307, ompTϪ had three characteristics that made it HhaI restriction fragment length polymorphism. Additionally, restric- particularly suitable as a source for the purification of T7 gene 5 tion enzyme analysis was used to verify that the A45T fragment was protein. First, its thioredoxin gene was deleted (19) to enable the puri- inserted into the vector fragment in the correct orientation and to verify fication of gene 5 protein alone, unbound to thioredoxin (12). Second, that the cloning procedure did not generate unwanted mutations at the this strain harbored a mutation in the OmpT endoprotease, which ligation junctions. Plasmid pGP5–5-A45T was then transformed into E. proteolytically cleaves T7 RNA polymerase during purification (38). coli A307, ompT to generate the strain E. coli A307, ompT(pGP5–5- by guest on October 5, 2019 Recent work suggested that the OmpT protease may also cleave the WT A45T), and the A45T gene 5 protein was purified from this strain. 2 T7 gene 5 protein (39) during purification. Third, this strain was a The expression and purification of the mutant A45Tg5P were per- HfrC strain and was sensitive to phage M13 infection. This third trait formed by the same procedure described for the E319Kg5P. A 2-liter was required, because the overexpression of the E319K gene 5 was culture of E. coli A307, ompT(pGP5–5-A45T) cells was grown, and5gof induced by infection with phage M13mGP1–2 in the presence of isopro- cells (wet weight) were harvested. The purification of the A45Tg5P from pyl-␤-D-thiogalactopyranoside (25). this5gofcells is summarized in Table I. A45Tg5P was purified The E319Kg5P was purified from E. coli A307, ompT(pGP5–5- approximately 700-fold with a total yield of 12%. Fraction IV was E319K) using a method that deviated slightly from a previously de- estimated to be 90% pure, as judged by denaturing polyacrylamide gel scribed procedure (8). Bacteriophage M13mGP1–2 was propagated by electrophoresis. infecting E. coli MV1190 and was purified as described (24). Five liters DNA Polymerase Assays—DNA polymerase activity was measured of E. coli A307, ompT(pGP5–5-E319K) cells were grown with vigorous by a modification of previous procedures (4, 8). DNA polymerase assays aeration and stirring (350 rpm) in a Magnaferm fermentor (New Brun- were performed using either heat-denatured (4) calf thymus DNA (Sig- swick Scientific Co., Inc.) in rich media (2% tryptone, 1% yeast extract, ma) or singly primed M13mp18 DNA as a template. Primed M13mp18 0.5% NaCl, 0.2% dextrose, 0.2% casamino acids, 10 mM NaOH, 20 mM DNA was made by annealing (8) an oligodeoxynucleotide (5Ј-GTTTTC- KPO , pH 7.4) at 37 °C. At a cellular concentration of approximately 109 4 CCAGTCACGAC-3Ј from U.S. Biochemical Corp.) to M13mp18 DNA cells/ml (A ϭ 4), aeration was reduced, and the speed of the stirring 590 (U.S. Biochemical Corp.). drive was lowered to 100 rpm. These alterations in culture conditions DNA polymerase activity measured the amount of [3H]dTMP incor- were necessary to allow the cells to generate pili, required for M13 porated into DNA during incubation at 37 °C in a buffer containing 2 nM attachment. After 30 min of cell growth in conditions of low aeration gene 5 protein, 2 ␮M thioredoxin, 300 ␮M of each dNTP (dATP, dCTP, and stirring, the temperature of the culture was lowered to 34 °C, and dGTP, and [3H]dTTP to a specific activity of approximately 5 cpm/ the overexpression of E319Kg5P was induced by the addition of 0.5 mM pmol), 50 mM NaCl, 10 mM MgCl ,40mM Tris-Cl, pH 7.5, 5 mM isopropyl-␤-D-thiogalactopyranoside and M13mGP1–2 at a multiplicity 2 dithiothreitol, and either 100 ␮g/ml heat-denatured calf thymus DNA of infection of 50. After another 30 min to allow for phage adsorption or 20 nM primed M13mp18 DNA as a template. Reactions were termi- and penetration, the amount of aeration and stirring was returned to nated by adding EDTA to a final concentration of 50 mM, and the initial levels. The cells were harvested (35 g wet weight) 2.5 h later as reaction mixture was then placed on DE81 ion exchange paper. Unin- described (8). Purification of the mutant E319Kg5P was as described corporated nucleotides were removed by washing the paper in a solu- previously (8), but the last purification step (hydroxyapatite chroma- tion of 500 mM Na HPO , pH 7.0 (28), at 25 °C for 1 h, with a change tography) was omitted. The purification of the E319K gene 5 protein is 2 4 summarized in Table I. The mutant E319K gene 5 protein was purified into fresh washing solution every 20 min. The washed paper was dried approximately 50-fold from the crude extract with a 4% total yield. under a heat lamp, and the amount of radioactivity bound to the paper Fraction V was greater than 90% pure, as estimated by denaturing was measured by liquid scintillation counting. Under these conditions, polyacrylamide gel electrophoresis followed by silver staining. a unit of DNA polymerase activity was defined as the amount of enzyme Cloning, Expression, and Purification of the Ala-45 to Thr-45 (A45T) required to catalyze the incorporation of 10 nmol of deoxynucleotides Gene 5 Protein—Cloning and purification of the A45Tg5P were accom- into DNA at 37 °C in 30 min. plished in a manner analogous to that for the E319Kg5P. DNA from the Exonuclease Assays—3Ј to 5Ј exonuclease activity was measured by modifying previously described procedures (36, 40). Reaction conditions for the exonuclease assay contained 40 mM Tris-Cl, pH 7.5, 10 mM 2 S. Tabor, personal communication. MgCl2,5mMdithiothreitol, 50 mM KCl, 1500 pmol (in nucleotide equiv- 20002 T7 DNA Polymerase-E. coli Thioredoxin Interaction alents) of double-stranded (6 nM) or single-stranded (12 nM)[3H]DNA, 3 TABLE II nM gene 5 protein, and, when present, 1 ␮M thioredoxin. The reaction DNA polymerase activity of the various gene 5 mixture was incubated at 37 °C in a total volume of 100 ␮l, and the protein-thioredoxin complexes reactions were terminated by adding EDTA to a final concentration of DNA polymerase activity was measured using primed M13mp18 as a 50 mM. DNA was then precipitated by adding 30 ␮l of an ice-cold template as described under “Experimental Procedures.” Specific activ- solution of bovine serum albumin (10 mg/ml) and 30 ␮l of an ice-cold ity of DNA polymerase was defined as pmol of deoxynucleotide incor- solution of trichloroacetic acid (100% w/v). After a 20-min incubation on porated into DNA per ng of protein per min. The activity values in this ice, the precipitated DNA was removed by centrifugation at 12,000 ϫ g table are average values from three experiments. S.D. of the numerical for 20 min at 4 °C. The amount of radioactivity in 125 ␮l of the resulting sample is shown in parentheses. supernatant was measured by liquid scintillation counting using 6 ml of Gene 5 protein-thioredoxin complex Specific activity (S.D.) Ultima Gold (Packard Instrument Co.) as a fluor. Double-stranded [3H]DNA was synthesized by PCR. The reaction WT-WT 20 (1) mixture (100 ␮l) contained 20 mM Tris-Cl, pH 8.3 at 20 °C, 1.5 mM WT-G74D 3 (1) E319K-WT 74 (11) MgCl ,25mMKCl, 0.05% Tween 20, 100 ␮g/ml bovine serum albumin, 2 E319K-G74D 51 (2) 30 ␮M of each dNTP (dATP, dCTP, dGTP, and dTTP), 5 units of Am- A45T-WT 56 (2) pliTaq DNA polymerase, 20 pmol of each primer, approximately 106 A45T-G74D 8 (3) template molecules, and 10 ␮lof[3H]dTTP (43 Ci/mmol, 1 mCi/ml). The amplified product (a 1200-base pair, linear DNA fragment) was purified 3 by the Geneclean kit. The specific activity of the [ H]DNA was typically RESULTS between 10 and 50 cpm/pmol in nucleotide equivalents. Single-stranded [3H]DNA was prepared by heating the labeled double-stranded DNA at We (1) developed a genetically based method to probe the 100 °C for 5 min, followed by immediate chilling on ice. interactions between the bacteriophage T7 DNA polymerase, Measuring Binding Between T7 Gene 5 Protein and E. coli Thiore- product of T7 gene 5, and its processivity factor, E. coli thioredoxin—The binding affinity between gene 5 protein and thioredoxin doxin. We used an E. coli strain harboring a Gly-74 to Asp-74 was measured as described previously (36). Gene 5 protein and thiore- (G74D) thioredoxin mutation that was unable to support WT

doxin, purified separately, were reconstituted in solution, and produc- Downloaded from T7 growth to select for suppressor T7 strains that contained tive binding was monitored by measuring DNA polymerase activity. The computer program KaleidaGraph (developed by Abelbeck Soft- compensatory mutations in its gene 5. Six different mutations ware) was used to construct a Scatchard plot ([bound] on the ordinate in T7 gene 5 are individually necessary and sufficient to com- versus [bound]/[free] on the abscissa) from data generated by the DNA pensate for the G74D thioredoxin defect. Three suppressor polymerase activity assay. To represent DNA polymerase activity in a mutations (E319K, E319V, and Y409C) reside within the po- Scatchard plot format, [bound] was estimated to be the amount of DNA lymerization domain of T7 DNA polymerase, and the other synthesized, and when thioredoxin was added in large molar excess http://www.jbc.org/ three suppressor mutations (V3I, V32A, and A45T) reside over gene 5 protein, [free] was estimated to be the total concentration of thioredoxin in the reaction (36). The program was also used to generate within the 3Ј to 5Ј exonuclease domain. In order to understand a line that best fitted the given data and to calculate the negative slope the mechanism of suppression, we have biochemically charac- of that line, the observed equilibrium dissociation constant (Kobs) be- terized the G74D thioredoxin and two of the mutant gene 5 tween gene 5 protein and thioredoxin (36). KaleidaGraph was also used proteins, E319K and A45T, representatives of suppressor mu- to calculate a correlation coefficient (r) that measures how well the tations in the polymerase and exonuclease domains, respec- given data fit the generated line. A correlation coefficient of 1 (r ϭ 1) tively. G74D thioredoxin (G74DTrxA), E319K gene 5 protein by guest on October 5, 2019 would mean that the data produced a line perfectly. Processivity Assays—Processivity of polymerization was measured (E319Kg5P), and A45T gene 5 protein (A45Tg5P) were purified by a modification of a previously described procedure (8). An oligode- to apparent homogeneity from E. coli cells overexpressing the oxynucleotide (5Ј-GTTTTCCCAGTCACGAC-3Ј) complementary to mutant genes as described under “Experimental Procedures.” M13mp18 DNA was labeled with 32P at its 5Ј-end using T4 polynucle- DNA Polymerase Activity—We began our biochemical char- 32 otide kinase. The P-5Ј-end-labeled oligonucleotide was annealed to acterization by reconstituting the three gene 5 proteins (WT, M13mp18 DNA (8), and the 5Ј-labeled primed M13mp18 DNA was E319K, and A45T) with the two thioredoxins (WT and G74D) in purified by using the Geneclean kit. DNA synthesis was carried out in all six possible combinations and by measuring the DNA po- a reaction that contained 300 ␮M of each dNTP (dATP, dCTP, dGTP, lymerase activity of each gene 5 protein-thioredoxin complex. and dTTP), 40 mM Tris-Cl, pH 7.5, 10 mM MgCl2,50mM NaCl, 5 mM dithiothreitol, 20 nM 5Ј-labeled primed M13mp18 DNA, 2 nM gene 5 These experiments were performed to compare quantitatively protein, and 2 ␮M thioredoxin. The reaction mix was preincubated for a the activities of the various mutant complexes and also to minute at 37 °C without proteins, and the reaction was started by the evaluate qualitatively the interaction between a particular addition of the proteins. At various times after the start, the reaction gene 5 protein and a corresponding thioredoxin. The results are was terminated by adding EDTA to a final concentration of 50 mM, and the products of the reaction were analyzed by electrophoresis through a summarized in Table II. 0.8% agarose gel containing ethidium bromide (0.5 ␮g/ml). The gel was Both mutant gene 5 proteins, when complexed with the then dried, and the location of the radioactivity was determined by mutant G74D thioredoxin, were more active (3-fold for the autoradiography. A45Tg5P and 17-fold for the E319Kg5P) than WT gene 5 pro- Molecular Structures—The computer graphics program RasMol ver- tein complexed to the same mutant thioredoxin. This result 3 sion 2.5 was used to visualize protein and protein-DNA structures. The was expected since the A45T and E319K mutations in gene 5 structure of reduced thioredoxin from E. coli (41) and the structure of were isolated as suppressors of the G74D thioredoxin mutation the large Klenow fragment of E. coli DNA polymerase I complexed with DNA (42) were obtained through the internet from the Protein Data (1). From the data in Table II, it is evident that the A45T and Bank (Brookhaven National Laboratories).4 With the permission of Dr. E319K mutations are not allele-specific for DNA polymerase Dyson, the atomic coordinates for thioredoxin were used to generate activity in vitro, because both mutant gene 5 proteins are able Fig. 3, and similarly, with the permission of Dr. Beese, the atomic to form an active DNA polymerase when complexed to either coordinates for the E. coli DNA polymerase I-DNA complex were used to the mutant thioredoxin or the WT thioredoxin. In fact, both generate Fig. 4. Fig. 5 was created by merging and manipulating Figs. mutant gene 5 proteins had more polymerase activity when 3 and 4, using the computer program Canvas 3.5 (Deneba Software). complexed to the WT thioredoxin than when complexed to the mutant thioredoxin. This in vitro result is consistent with the in vivo results since E. coli strains encoding WT thioredoxin are 3 Program written by Roger Sayle (Biomolecular Structures Group, able to support the growth of both suppressor phage strains, Glaxo Research and Development, Greenford, United Kingdom), ob- T7–5-A45T and T7–5-E319K. tained free of charge by anonymous FTP (file transfer protocol) from ftp.dcs.ed.ac.uk. Binding Affinity—To quantitatively assess the interaction 4 Obtained from http://www.pdb.bnl.gov. between a given gene 5 protein and a particular thioredoxin, T7 DNA Polymerase-E. coli Thioredoxin Interaction 20003

TABLE III Binding affinity between gene 5 proteins and thioredoxins

Kobs (the observed equilibrium dissociation constant) and r (correlation coefficient) were calculated from Fig. 1, A and B, as described under “Experimental Procedures.” The values in the table are average values from four experiments, and S.D. values are shown in parentheses. r is a quantitative estimate of how well the data fit a given line.

Gene 5 protein-thioredoxin Kobs (S.D.) r (S.D.)

␮M WT-WT 0.4 (0.07) 0.90 (0.02) E319K-WT 0.3 (0.16) 0.97 (0.03) A45T-WT 0.6 (0.2) 0.92 (0.06) WT-G74D 12.9 (2.3) 0.98 (0.02) E319K-G74D 2.4 (0.5) 0.99 (0.01) A45T-G74D 20.3 (4.5) 0.91 (0.09)

mutation, is reduced to a 6-fold increase (0.4 ␮M to 2.4 ␮M)by the compensating E319K mutation in gene 5. It is also evident that the A45T mutation in gene 5 protein does not restore binding to the G74D thioredoxin; in fact, the A45Tg5P bound to

the G74D thioredoxin with less affinity (Kobs ϭ 20.3 ␮M) than the binding of the WTg5P to the G74D thioredoxin (12.9 ␮M). This result was puzzling since the A45T mutation in gene 5 protein was isolated as a suppressor mutation that compen- Downloaded from sated in vivo for the G74D thioredoxin defect. Evidently, the A45Tg5P mutation is suppressing the G74D thioredoxin mutation by a more complicated mechanism. Since the A45T mutation is located within the putative 3Ј to 5Ј exonuclease domain of T7 gene 5 protein (1, 40), the 3Ј to 5Ј exonuclease activity of all the proteins was evaluated (see below). http://www.jbc.org/ It should be noted that Huber et al. (36), using the same procedure, also measured the binding affinity between WT T7 gene 5 protein and WT thioredoxin (36). As shown in Table III,

we calculated the Kobs of the interaction between WT gene 5 protein and WT thioredoxin to be 400 nM at 37 °C. In contrast, by guest on October 5, 2019 Huber et al. (36) reported values of 5 nM at 30 °C and 25 nM at 42 °C for the same interaction. We attribute this approximately 20-fold difference in binding affinity primarily to differences in template and protein preparation, both of which can potentially affect the results quite significantly. To circumvent these prob- lems, all measurements reported in this work were made using FIG.1. Scatchard plots of the binding of WTg5P, E319Kg5P, identical template and buffer preparations to ensure internal and A45Tg5P to WT thioredoxin (A) or mutant G74D thioredoxin relative consistency throughout the course of this study. (B). The purified gene 5 proteins (WT, E319K, and A45T) were mixed Exonuclease Assays—The 3Ј to 5Ј exonuclease activity of all with increasing concentrations of the WT (A) or G74D (B) thioredoxin in a DNA polymerase activity assay. The amount of DNA synthesis was six T7 gene 5 protein-thioredoxin complexes was determined, measured as described under “Experimental Procedures,” and the data and the results are summarized in Table IV. As expected, the were used to generate a Scatchard plot. The observed equilibrium A45Tg5P, harboring a mutation in the putative exonuclease dissociation constant (K ) for a particular gene 5 protein-thioredoxin obs domain of the protein, had reduced exonuclease activity (11%) complex was the negative slope of the line that best fitted the data. These data were generated from a single experiment. This same exper- as compared with the WT activity with single-stranded DNA as iment was repeated four times, and the combined data are summarized a substrate. Even when complexed to thioredoxin, the A45Tg5P in Table III. had reduced exonuclease activity as compared with the WTg5P. With single-stranded DNA as a substrate, the E319Kg5P also the observed equilibrium dissociation constant (Kobs) between had reduced exonuclease activity (28%) as compared with the each gene 5 protein and each thioredoxin was determined as WT activity. However, when complexed with the WT thiore- described previously (36). The results of binding assays be- doxin, the E319Kg5P had only slightly less activity (84%) than tween the three gene 5 proteins (A45T, E319K, and WT) and the WTg5P-WTTrxA complex, and when bound to the mutant WT E. coli thioredoxin are presented in Fig. 1A. The results of G74D thioredoxin, the E319Kg5P had approximately 5-fold binding studies between the three gene 5 proteins and the more activity than the WTg5P bound to the same mutant mutant G74D thioredoxin are shown in Fig. 1B. The salient thioredoxin. As with the results of the binding assay, this last features of the binding studies are summarized in Table III. result suggests that the E319K mutation restored the interac- From inspection of Fig. 1, A and B, and Table III, it is evident tion with the G74D thioredoxin. that the E319K mutation in gene 5 protein restored, by approx- The 3Ј to 5Ј exonuclease activities of the three gene 5 pro- imately 80%, the binding defect created by the G74D mutation teins (uncomplexed to thioredoxin) with double-stranded DNA in thioredoxin. The Kobs of the WTg5P-WTTrxA interaction (0.4 as substrate are not shown, because gene 5 protein alone has ␮M) is increased by approximately 32-fold to 12.9 ␮M for the negligible exonuclease activity on double-stranded DNA sub-

WTg5P-G74DTrxA interaction, and the Kobs of the E319Kg5P- strates (7, 9). Similarly, the exonuclease activities of the vari- G74DTrxA interaction is 2.4 ␮M. Therefore, the 32-fold increase ous gene 5 protein-thioredoxin complexes on a single-stranded

(0.4 ␮M to 12.9 ␮M)intheKobs, caused by the G74D thioredoxin DNA substrate are not shown, since thioredoxin stimulates 20004 T7 DNA Polymerase-E. coli Thioredoxin Interaction

TABLE IV 3Ј to 5Ј exonuclease activity of the WT and mutant proteins 3Ј to 5Ј exonuclease activity was measured as described under “Ex- perimental Procedures.” The exonuclease activities were evaluated in three assay systems: (a) the three gene 5 proteins with single-stranded DNA (ssDNA) as a substrate, (b) the gene 5 proteins complexed to the WT thioredoxin with double-stranded DNA (dsDNA) as substrate, and (c) the gene 5 proteins complexed to the G74D thioredoxin with double- stranded DNA as substrate. All activities are presented as a percentage of the WT gene 5 protein activity. The values are derived from average values of three experiments. The standard deviations are shown in parentheses.

dsDNA dsDNA Gene 5 protein-thioredoxin ssDNA (WTTrxA) (G74DTrxA) WTg5P 100a E319Kg5P 28 (3) A45Tg5P 11 (1) WTg5P-WTTrxA 100a E319Kg5P-WTTrxA 84 (16) A45Tg5P-WTTrxA 70 (8) FIG.2.Processivity of polymerization. The processivity of polym- WTg5P-G74DTrxA 100a erization was assayed as described under “Experimental Procedures.” E319Kg5P-G74DTrxA 517 (121) Each gene 5 protein-thioredoxin complex was allowed to synthesize A45Tg5P-G74DTrxA 44 (1) DNA from a primed M13mp18 template with a 5Ј-32P-label on the a To compare the exonuclease activities between complexes with the primer. For each polymerase complex, the reaction was terminated at 1, WT and with the mutant thioredoxin, it should be noted that the 5, and 15 min. The products of the reaction were separated electro-

WTg5P-WTTrxA complex has about 20-fold more activity than the phoretically through an agarose gel, and the gel was then dried and Downloaded from WTg5P-G74DTrxA complex with double-stranded DNA as a substrate. autoradiographed. Lanes 1–3 were produced from reactions that con- The exonuclease activity of WTg5P with single-stranded DNA as a tained the WTg5P-WTTrxA complex. Lane 1 was generated from a substrate was roughly half of the activity of the WTg5P-WTTrxA com- reaction that was terminated at 1 min. Lane 2 was terminated at 5 min, plex with double-stranded DNA as a substrate. and lane 3 was terminated at 15 min. Similarly, lanes 4–6 contained the E319Kg5P-WTTrxA complex that was terminated at 1, 5, and 15 min, respectively. Lanes 7–9 contained the A45Tg5P-WTTrxA complex only slightly the exonuclease activity of gene 5 protein on

at 1, 5, and 15 min. Lanes 10–12 contained the WTg5P-G74DTrxA http://www.jbc.org/ single-stranded substrates (7, 9). complex at 1, 5, and 15 min. Lanes 13–15 contained the E319Kg5P- Processivity of Polymerization—The ability of thioredoxin to G74DTrxA complex at 1, 5, and 15 min, and lanes 16–18 contained the stimulate the DNA polymerase activity of T7 gene 5 protein (6, A45Tg5P-G74DTrxA complex at 1, 5, and 15 min. The mobility of the primed M13mp18 template, without protein, is indicated, and the po- 7) is due primarily to an increase in the processivity of nucle- sitions of size markers (linear, double-stranded DNA) are also otide polymerization by gene 5 protein (8, 14). Therefore, the indicated. processivity of each gene 5 protein-thioredoxin complex was by guest on October 5, 2019 examined to determine if the gene 5 and thioredoxin mutations should be more DNA synthesis in lanes 16–18 as compared (E319K and A45T in gene 5 and G74D in thioredoxin) affected with lanes 10–12. However, there is no obvious difference be- the ability of thioredoxin to confer processivity upon gene 5 tween lanes 10–12 and lanes 16–18, suggesting that the A45T protein. Processivity of nucleotide polymerization was exam- mutation in gene 5 protein does not suppress the G74D thiore- 32 ined as described under “Experimental Procedures.” A P-5Ј- doxin defect by increasing the processivity of nucleotide polym- end-labeled oligodeoxynucleotide was annealed to single- erization. This experiment was repeated several times under stranded M13mp18 DNA, and DNA synthesis was carried out slightly different conditions, and in each case, no difference in using a 10:1 molar ratio of primer-template to enzyme (gene 5 processive DNA synthesis between the A45Tg5P-G74DTrxA protein-thioredoxin). For each gene 5 protein-thioredoxin com- complex and the WTg5P-G74DTrxA complex could be detected. plex, the polymerization reaction was terminated at 1, 5, and Burst Sizes of Phages T7 WT, T7–5-A45T, and T7–5- 15 min, and the reaction products were examined by electro- E319K—In order to compare the in vitro data with the in vivo phoretic separation through an agarose gel. The results are phenotypes, we measured the burst sizes of the WT T7 phage, shown in Fig. 2. phage T7–5-A45T, and phage T7–5-E319K (Table V). In com- A comparison of lanes 1–3 and lanes 10–12 shows that the parison with T7–5-E319K, T7–5-A45T grew poorly within E. G74D thioredoxin mutation decreases the ability of thioredoxin coli cells encoding the G74D thioredoxin mutation and also to confer processivity upon the WT gene 5 protein. In fact, in within WT E. coli cells. As expected, strain T7–5-E319K was this assay, processive nucleotide polymerization by the WTg5P- able to grow well within the E. coli strain harboring the G74D G74DTrxA complex is undetectable (lanes 10–12). A compari- thioredoxin mutation. Consistent with the in vitro binding son of lanes 1–3 and lanes 4–6 shows that the E319K mutation studies, the E319K mutation in gene 5 protein did not restore in gene 5 does not significantly alter the ability of gene 5 the interaction with the G74D thioredoxin to a full extent, since protein to interact with the WT thioredoxin; however, as ex- the burst size of T7–5-E319K within the G74D thioredoxin host pected, the E319K mutation in gene 5 does increase the ability was 36% (39⁄109) of the burst size of the WT T7 within the WT to interact with the G74D mutant thioredoxin (compare lanes host. In contrast to both suppressor phage strains, WT T7 was 10-12 with lanes 13–15). not able to grow within the E. coli strain that encoded the G74D Since the A45T mutation in gene 5 reduced the 3Ј to 5Ј thioredoxin mutation. exonuclease activity (Table IV) and did not restore binding to the G74D thioredoxin (Table III), we considered the possibility DISCUSSION that it was compensating for the thioredoxin defect by altering Previously, we found that a G74D thioredoxin mutant of E. the processivity of nucleotide polymerization. A comparison of coli was unable to support the growth of WT T7 phage, and we lanes 10–12 and lanes 16–18 of Fig. 2 shows that the A45Tg5P have used this mutant strain to select for compensatory extra- does not detectably increase the processivity of polymerization genic suppressors in T7 gene 5 protein (1). We found that the in this assay. If the A45Tg5P-G74DTrxA complex was more G74D thioredoxin mutant was compensated in vivo by six processive than the WTg5P-G74DTrxA complex, then there different alterations in gene 5 protein, each of which individu- T7 DNA Polymerase-E. coli Thioredoxin Interaction 20005

TABLE V ity of polymerization (Fig. 2). The results of the in vivo burst Burst sizes of phage strains T7 WT, T7–5-A45T, and T7–5-E319K size studies (Table V) are consistent with the in vitro results, Burst sizes were determined as described under “Experimental Pro- since the strain T7–5-A45T grew very poorly within E. coli cells cedures.” The values in the table are averages of three experiments. harboring the G74D thioredoxin defect. Based on these results, T7 phage strain Thioredoxin in host E. coli strain Burst size we suggest that the alanine 45 residue of T7 gene 5 protein is WT WT 109 not physically adjacent to the glycine 74 residue of thioredoxin 5-A45T WT 1–5 during T7 DNA replication within infected cells. At present, we 5-E319K WT 80 are unable to identify the nature of this compensating effect, WT G74D 0 5-A45T G74D 1–5 but it could be due to the interaction of the A45Tg5P- 5-E319K G74D 39 G74DTrxA complex with other proteins at the T7 replication fork. Based upon these functional studies, we have attempted to ally was both necessary and sufficient for suppression of the build a model of the gene 5 protein-thioredoxin interaction. thioredoxin defect. Three of the suppressor mutations (V3I, Necessarily, the focus of this model revolves around the iden- V32A, and A45T) were located within the 3Ј to 5Ј exonuclease tification of regions or surfaces of contact between the two domain of gene 5 protein, and the remaining three (E319V, proteins. Fortunately, we were aided greatly in developing our E319K, Y409C) were within the polymerization domain of gene model by the work of others. Dyson et al. (41) determined by 5 protein. nuclear magnetic resonance spectroscopy the solution struc- In this study, we have extended our initial genetic analysis of ture of reduced thioredoxin, the conformation of thioredoxin the gene 5 protein-thioredoxin interaction with a biochemical that binds productively to T7 gene 5 protein (43). Eklund et al. analysis of the same interaction. Specifically, we have overex- (44) had suggested previously that a flat, hydrophobic surface pressed and purified the G74D mutant thioredoxin, as well as surrounding the active site cysteines (Cys-32 and Cys-35) of Downloaded from two suppressor gene 5 proteins (E319K and A45T). These mu- thioredoxin plays a particularly significant role in interactions tant gene 5 proteins were chosen to represent both classes of with other proteins. Russel and Model (45), investigating the suppressor mutations, one (E319K) from the polymerization requirement for E. coli thioredoxin in the assembly of the domain of gene 5 protein and one (A45T) from the exonuclease filamentous bacteriophage f1, mutagenized the entire thiore- domain. Using the purified proteins, we have examined their doxin gene by nitrosoguanidine and isolated three missense interactions, and based upon our results, we have constructed

mutations within thioredoxin (G74D, G92S, and G92D) that http://www.jbc.org/ a model of the gene 5 protein-thioredoxin interaction. caused an inability to support growth of WT f1 phage (19). We believe that the E319K suppressor mutation in T7 gene Supporting the hypothesis put forth by Eklund et al. (44), both 5 protein compensates for the G74D thioredoxin alteration the Gly-74 and Gly-92 residues are located within the flat, primarily by restoring the binding affinity. We propose this hydrophobic, putative interacting surface. As previously estab- mechanism of suppression for several reasons. First, the data lished (1), a G92D thioredoxin mutant of E. coli does not sup- presented in Table III indicate that the E319K mutation report the growth of WT T7 phage, and in contrast to phage f1, a by guest on October 5, 2019 stored the binding affinity to the G74D thioredoxin by approx- G92S thioredoxin mutant is able to support WT T7 growth imately 80%. Second, the data presented in Table IV show that (data not shown). Krause and Holmgren (46) found that replac- the E319K mutation in gene 5 protein does not cause a signif- ing the highly conserved Trp-31 residue (adjacent to the active icant alteration in the 3Ј to 5Ј exonuclease activity. Third, lanes 13–15 of Fig. 2 indicate that the E319K gene 5 protein is able site Cys-32) of thioredoxin with an alanine or a histidine resi- to interact with the G74D thioredoxin to form an efficient DNA due greatly reduced productive interaction with WT T7 gene 5 polymerase. Fourth, the burst size studies (Table V) indicate protein. Minarik et al. (47) found that an E. coli thioredoxin that T7–5-E319K grows quite well within the E. coli host mutant strain that replaced the conserved Gly-33 residue with harboring the G74D thioredoxin mutation. a tryptophan residue was unable to support the growth of WT Based upon these data, we conjecture that the Glu-319 res- T7 phage. idue of gene 5 protein and the Gly-74 residue of thioredoxin Other investigators (43, 48) have also identified this hydro- define a contact point between the two proteins. Specifically, phobic surface of thioredoxin (residues 31–35, 74–77, and 90– we suggest that, during phage T7 DNA replication in vivo, the 93) as critical for interactions with T7 gene 5 protein. In par- side chain atoms of glutamate 319 of T7 gene 5 protein are ticular, Adler and Modrich (43) found that alkylation of free physically adjacent to the side chain hydrogen atom of glycine reduced thioredoxin at Cys-32 and Cys-35 by exposure to chem- 74 of thioredoxin. When the Gly-74 of thioredoxin is replaced by icals that preferentially alter sulfhydryl moieties prevented the an aspartic acid residue, the juxtaposition of the negatively ability of thioredoxin to stimulate the DNA polymerase activity charged carboxyl groups of Glu-319 (g5P) and of Asp-74 (TrxA) of T7 gene 5 protein. Furthermore, T7 gene 5 protein, when generates ionic repulsion, resulting in the inability of the two associated with thioredoxin prior to exposure to the same al- proteins to associate productively. When the Glu-319 of gene 5 kylating agents, protected the inactivation of thioredoxin. Fi- protein is substituted with a positively charged lysine residue, nally, other investigators have determined the solution struc- repulsion due to the physical proximity of two negative charges ture of the reduced and oxidized thioredoxin and have is removed and perhaps is replaced by ionic attraction, restor- compared the two structures (49, 50). The two forms of thiore- ing the ability to associate productively with the Asp-74 doxin are extremely similar, essentially identical in backbone thioredoxin. structure. The authors further suggest that the functional dif- In contrast to the E319K mutation and as evident from ferences could perhaps be explained by differences in the con- inspection of Table III, the mechanism of suppression for the formational flexibility of the region surrounding the active site, A45T mutation within gene 5 protein does not involve a direct especially residues 73–76. In reduced thioredoxin, the active restoration of binding affinity. The A45T mutation, located site region was found to be more dynamic or flexible than the within the putative 3Ј to 5Ј exonuclease domain of gene 5 same region in oxidized thioredoxin. Presumably, this in- protein, did reduce the exonucleolytic activity of the protein creased conformational flexibility in the hydrophobic region (Table IV); however, the reduction in exonucleolytic activity surrounding the active site facilitates interactions with other was not accompanied by a detectable increase in the processiv- proteins. 20006 T7 DNA Polymerase-E. coli Thioredoxin Interaction

FIG.3.A surface of reduced thioredoxin that interacts with T7 gene 5 protein. The solution structure of reduced thioredoxin, determined by nuclear magnetic resonance spectroscopy (41), was used to generate this figure as described under “Experimental Procedures.” In this illustration, five amino acid residues (Trp-31, Cys-32, Cys-35, Gly- 74, and Gly-92) are shown in a space-filling model, and the remainder of the molecule is represented by its backbone only, without the side- chain atoms. Alterations in any of these five amino acid residues can cause a reduction in the ability to interact productively with T7 gene 5 protein (1, 43, 46). In this figure, nitrogen atoms are purple, and carbon FIG.4.A model for the structure of T7 gene 5 protein bound to atoms are gray. Oxygen atoms are red, and sulfur atoms are yellow. The DNA based on the structure of an E. coli DNA polymerase I hydrogen atoms of residues Trp-31 (W31), Cys-32 (C32), Cys-35 (C35), Klenow fragment-DNA complex. The structure of the Klenow frag- and Gly-92 (G92) are white, and for emphasis, the hydrogen atoms of ment of E. coli DNA polymerase I bound to DNA was determined by Gly-74 (G74) are green. In the G74D mutant, an aspartate moiety x-ray crystallography (42). The DNA is dark blue, and the protein (CH COOϪ) would replace one of the green side-chain hydrogen atoms. backbone (devoid of side-chain atoms) is gray. The positions of six amino Downloaded from 2 acid residues are shown in three different colors. Phe-762 (F762), very near the site of DNA polymerization (63), is green. Based upon the Based upon the arguments presented above, we speculate homology between E. coli DNA polymerase I and T7 gene 5 protein (51, that residues 32–35, 73–77, and 90–93 of thioredoxin, the 52, 54, 64), tertiary structure inference was used to map or to position hydrophobic region first emphasized by Eklund et al. (44), T7 gene 5 protein residues on the correspondingly homologous residues defines a surface of thioredoxin that interacts with T7 gene 5 of E. coli DNA polymerase I. Four amino acid residues, indicating the protein. Fig. 3 shows the solution structure of reduced thiore- positions of four different T7 gene 5 suppressor mutations that com- http://www.jbc.org/ pensate for the G74D thioredoxin defect (1), are colored cyan. These four doxin (41) and illustrates our hypothesis. Since an aspartate amino acids are labeled with the E. coli DNA polymerase I residue residue, instead of a glycine residue, at either position 74 or 92 followed by the corresponding T7 gene 5 protein residue, with a slash destroys the ability of thioredoxin to interact with T7 gene 5 between the two residues. The suppressor or compensatory substitu- protein (1) and since alkylation of the active site cysteines also tions are also indicated in the label. For example, the label L404/A45T identifies this particular residue as leucine 404 of E. coli DNA polym- inhibits binding to gene 5 protein (43), the active site cysteines, erase I, and this residue corresponds to alanine 45 of T7 gene 5 protein, by guest on October 5, 2019 Gly-74, and Gly-92 define part of the thioredoxin surface that is which, when substituted with threonine, compensates in vivo for the likely to contact gene 5 protein. In the G74D thioredoxin mu- G74D thioredoxin mutation. The His-571 residue is red, and the puta- tant, an aspartic acid side chain (–CH COOϪ) would presum- tive thioredoxin binding domain of T7 gene 5 protein would be inserted 2 close to or at this residue of E. coli DNA polymerase I. ably extend outward from one of the side chain hydrogen atoms (Fig. 3, green) of the Gly-74 residue. Unlike E. coli thioredoxin, the structure of T7 gene 5 protein suppressor mutations are shown in Fig. 4. The six suppressor has not been determined. However, T7 gene 5 protein belongs mutations that were located at Glu-319 altered the glutamate to a family of DNA-dependent DNA polymerases that shares residue to either a lysine or a valine residue, and in this work, substantial amino acid sequence homology to E. coli DNA po- we have argued that the Glu-319 residue of gene 5 protein and lymerase I (51, 52), and fortunately, the structure of the large the Gly-74 residue of thioredoxin define a contact point be- Klenow fragment of E. coli DNA polymerase I has been deter- tween the two proteins. Similarly, we have postulated that the mined by x-ray crystallography (53). More recently, the struc- Ala-45 residue of gene 5 protein does not contact the Gly-74 ture of the Klenow fragment of E. coli DNA polymerase I residue of thioredoxin. We now propose that the roughly 65 complexed to duplex DNA has also been determined (42). By amino acids of T7 gene 5 protein (residues 260–325) encode a tertiary structure inference based upon sequence homology, we putative thioredoxin binding domain. We postulate that most propose to use the crystal structure of DNA polymerase I Kle- of the gene 5 protein residues that contact thioredoxin directly now fragment complexed to double-stranded DNA as a rudi- would be located within this domain, and we estimate that, in mentary model for T7 gene 5 protein bound to duplex DNA. By comparison to the Klenow fragment, this added domain would this homology-based inference, we were able to match the A45T be inserted into the protein sequence of E. coli DNA polymerase suppressor mutation in T7 gene 5 protein with its homologous I somewhere close to the His-571 residue (Fig. 4, red). counterpart in E. coli DNA polymerase I, Leu-404 (54). This is In the model shown in Fig. 5, we have combined the flat, illustrated in Fig. 4. hydrophobic surface of reduced thioredoxin with the putative Attempts to similarly match the Glu-319 residue of T7 gene thioredoxin binding domain of gene 5 protein to generate a 5 protein with the homologous residue of E. coli DNA polym- structural model of the T7 gene 5 protein-thioredoxin complex erase I were not so straightforward, because, amid the highly bound to duplex DNA. Our model is rudimentary and lacks the homologous sequences, there is a domain of T7 gene 5 protein necessary details that would be required to understand pre- (roughly 65 amino acid residues in length) that does not have a cisely how thioredoxin stabilizes the primer-template-DNA po- homologous counterpart in E. coli DNA polymerase I (52, 55). lymerase complex (14). Most likely, by an unknown mecha- The Glu-319 residue of T7 gene 5 protein is located within this nism, thioredoxin confers processivity by physically domain. Previously, we had isolated 10 mutant strains of T7 obstructing the otherwise rapid dissociation of the newly syn- that could grow within the G74D thioredoxin mutant of E. coli. thesized, double-stranded DNA from T7 gene 5 protein. Six of the suppressor mutations within gene 5 protein were Our structural model now serves as a testable, working located at Glu-319 (1), and the positions of the other four hypothesis. Several experiments have been performed in an T7 DNA Polymerase-E. coli Thioredoxin Interaction 20007

newly synthesized duplex DNA and not the single-stranded template strand during DNA synthesis. Recent work supports both of these predictions.5 Perhaps the ultimate test of our structural model must await the structural determination by x-ray crystallography of the T7 gene 5 protein-thioredoxin complex bound to double-stranded DNA.

Acknowledgments—We thank Anh Nguyen-Huynh for helping to transduce the ⌬trxA307 allele from E. coli JH20 into E. coli HMS231. We are grateful to Dr. Stanley Tabor for generously supplying WT thioredoxin, WT T7 gene 5 protein, plasmid pGP5–5, phage M13mGP1–2, and E. coli A307, ompT. We also thank Ben Beauchamp, Ella Bedford, Khandan Baradaran, and Stanley Tabor for critical read- ings of the manuscript.

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