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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 Amino Acid Residues Critical for the Interaction between Bacteriophage T7 DNA Polymerase and Escherichia coli Thioredoxin The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters 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 59 to 39 DNA polym- oredoxin define a contact point or site of interaction erase activity (6–8) and a 39 to 59 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 39 to 59 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 thiore- doxin 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 39 to 59 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 g 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 (DtrxA307,pcnB80), E. coli HMS231 (trxA1,pcnB1), 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 (F9,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 (F1.1A and F1.1B), were buffer containing 50 mM Tris-Cl, pH 8.2, 20 mM EDTA, 10% sucrose.
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