A Single-stranded DNA-binding Protein of Bacteriophage T7 Defective in DNA Annealing
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m303374200
ISSN1083-351X
AutoresLisa F. Rezende, Smaranda Willcox, Jack D. Griffith, Charles C. Richardson,
Tópico(s)Bacteriophages and microbial interactions
ResumoThe annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Δ26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy. The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Δ26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy. Gene 2.5 protein is required for bacteriophage T7 growth (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar). Gene 2.5 protein acts as a nonspecific single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; WT, wild-type; NTA, nitrilotriacetic acid; SSB protein, single-stranded binding protein.-binding protein, binding ssDNA preferentially over double-stranded DNA (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). ssDNA binding proteins participate in multiple steps of DNA replication, recombination, and repair (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar, 2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar, 3Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 4Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (448) Google Scholar, 5Reuben R.C. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1846-1850Crossref PubMed Scopus (49) Google Scholar, 6Scherzinger E. Litfin F. Jost E. Mol. Gen. Genet. 1973; 123: 247-262Crossref PubMed Scopus (40) Google Scholar, 7Araki H. Ogawa H. Virology. 1981; 111: 509-515Crossref PubMed Scopus (18) Google Scholar, 8Araki H. Ogawa H. Mol. Gen. Genet. 1981; 183: 66-73Crossref PubMed Scopus (15) Google Scholar, 9Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 10Kong D. Richardson C.C. EMBO J. 1996; 15: 2010-2019Crossref PubMed Scopus (52) Google Scholar, 11Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Lee J. Chastain 2nd, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 13Yu M. Masker W. J. Bacteriol. 2001; 183: 1862-1869Crossref PubMed Scopus (13) Google Scholar). Whereas gene 2.5 protein is functionally equivalent to Escherichia coli SSB protein and the bacteriophage T4 gene 32 protein, it lacks significant sequence homology to these proteins, and neither of these proteins can replace its function in vivo (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar). In addition, gene 2.5 protein binds ssDNA with a lower affinity than either the E. coli or T4 proteins (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). Gene 2.5 protein also physically and functionally interacts with T7 DNA polymerase and T7 gene 4 product, a primase/helicase (3Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 9Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 12Lee J. Chastain 2nd, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). These interactions are mediated by a highly acidic carboxyl-terminal motif and are essential for coordination of leading and lagging strand DNA synthesis in vitro (12Lee J. Chastain 2nd, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 14Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar, 15Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In addition to binding ssDNA and physically interacting with T7 DNA polymerase, gene 2.5 protein also facilitates the annealing of complementary strands of DNA (10Kong D. Richardson C.C. EMBO J. 1996; 15: 2010-2019Crossref PubMed Scopus (52) Google Scholar, 11Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar). Homologous DNA annealing is a vital activity during the process of DNA replication, recombination, and repair (17Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (144) Google Scholar). A number of proteins have evolved to carry out this vital function, such as the RecA protein (18Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar, 19Bianco P.R. Tracy R.B. Kowalczykowski S.C. Front. Biosci. 1998; 3: 570-603Crossref PubMed Google Scholar) and members of the single strand annealing family that includes the E. coli RecT protein, the Redβ protein from bacteriophage λ, and the eukaryotic annealing protein Rad52 (17Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (144) Google Scholar, 20Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (390) Google Scholar, 21Hall S.D. Kane M.F. Kolodner R.D. J. Bacteriol. 1993; 175: 277-287Crossref PubMed Google Scholar, 22Kmiec E. Holloman W.K. J. Biol. Chem. 1981; 256: 12636-12639Abstract Full Text PDF PubMed Google Scholar, 23Reddy G. Golub E.I. Radding C.M. Mutat. Res. 1997; 377: 53-59Crossref PubMed Scopus (85) Google Scholar). Unlike the RecA protein, the gene 2.5 protein does not require ATP (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar), and it cannot mediate strand transfer (11Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar). Gene 2.5 protein bears some similarity to the RecT protein and its family members, proteins that also mediate DNA annealing in an ATP-independent fashion (17Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (144) Google Scholar). Structurally, gene 2.5 protein differs from members of this family, which form multimeric ring structures in the presence and absence of ssDNA (24Kagawa W. Kurumizaka H. Ishitani R. Fukai S. Nureki O. Shibata T. Yokoyama S. Mol. Cell. 2002; 10: 359-371Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 25Singleton M.R. Wentzell L.M. Liu Y. West S.C. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13492-13497Crossref PubMed Scopus (176) Google Scholar, 26Passy S.I. Yu X. Li Z. Radding C.M. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4279-4284Crossref PubMed Scopus (84) Google Scholar). Gene 2.5 protein, on the other hand, is a dimer in solution (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar), and its three-dimensional structure resembles that of other ssDNA-binding proteins (27Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (83) Google Scholar). Similar to T4 gene 32 protein and E. coli SSB protein, gene 2.5 protein features an oligonucleotide/oligosaccaride binding fold (Fig. 1) (27Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (83) Google Scholar). Although both T4 gene 32 protein and E. coli SSB protein have been shown to mediate DNA annealing (28Christiansen C. Baldwin R.L. J. Mol. Biol. 1977; 115: 441-454Crossref PubMed Scopus (57) Google Scholar, 29Alberts B.M. Frey L. Nature. 1970; 227: 1313-1318Crossref PubMed Scopus (448) Google Scholar), T7 gene 2.5 protein does so much more efficiently (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar). The biochemical basis of the efficient DNA annealing activity of gene 2.5 protein is unknown. It seems likely that it involves interactions between two gene 2.5 protein-coated ssDNA molecules. A previous study has shown that the ability to bind ssDNA is critical for this reaction to occur (30Hyland E.M. Rezende L.F. Richardson C.C. J. Biol. Chem. 2003; 278: 7247-7256Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). It is also possible that interactions at the dimer interface are involved in this process. Two gene 2.5 proteins with alterations in the dimer interface retained the ability to mediate DNA annealing, in a manner similar to the WT protein, whereas a third did so in a slightly longer time period (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). We have recently employed a genetic screen to identify functional domains in gene 2.5 protein (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). One of the alterations uncovered by the screen mapped to a loop connecting the prominent α-helix to the β-barrel portion of the structure (Fig. 1). The exact residue, Arg-82, resides in a disordered region of the structure. Here we describe the purification and characterization of this protein and show that it is defective in DNA annealing. Bacterial Strains and Phage—E. coli HMS262 (F– hsdR pro leu – lac – thi – supE tonA – trxA –) and E. coli HMS 89 (xth1 thi – argE mtl – xyl – str – R ara – his – galK lacY proA leu – thr – tsx – supE) were used as hosts for phage experiments. E. coli BL21 (DE3) (F– ompT hsdSB(rB-mB-) gal – dcm – (DE3)) (Novagen) was used to express gene 2.5. T7Δ2.5 phage used in the in vivo DNA synthesis experiments was provided by Jaya Kumar (Harvard Medical School). Plasmids, Oligonucleotides, and Proteins—The following oligonucleotides were purchased from Oligos Etc.: T72.5NdeI (5′-CGTAGGATCCATATGGCTAAGAAGATTTTCACCTC-3′), T72.5BamH1 (5′-CGTAGGATCCACTTAGAAGTCTCCGTC-3′), and Oligo 70 (5′-GACCATATCCTCCACCCTCCCCAATATTGACCATCAACCCTTCAC CTCACTTCACTCCACTATACCACTC-3′). The oligonucleotide BCMP206 (5′-TAACGCCAGGGTTTTCCCAGTCACG-3′) was synthesized by the Biopolymer Laboratory, Harvard Medical School. M13 (mGP1-2) DNA and T7 DNA polymerase lacking exonuclease activity (30Hyland E.M. Rezende L.F. Richardson C.C. J. Biol. Chem. 2003; 278: 7247-7256Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) were kindly provided by Stanley Tabor (Harvard Medical School). Gene 2.5 protein-Δ26C was provided by Edel Hyland (Harvard Medical School). His-gene 2.5 protein-Δ26C was provided by James Stattel (Harvard Medical School). T7 DNA polymerase was provided by Donald Johnson and Joon-Soo Lee (Harvard Medical School). Purification of WT gene 2.5 protein and His-gene 2.5 protein was described previously (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). E. coli SSB protein was purchased from U.S. Biochemical Corp. All other proteins were purified as described below. In Vivo DNA Synthesis Assay—Phage DNA synthesis was determined as described previously (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). E. coli HMS262 cells transformed with pETGP2.5-R82C were grown in Davis minimal media supplemented with ampicillin at 30 °C. Cells were infected with T7Δ2.5 phage at a multiplicity of infection of 7. At 5-min intervals postinfection, 200-μl samples were removed. [3H]thymidine (50μCi/ml) was added, and after a 90-s incubation at 30 °C, 40 μl of an ice-cold solution of 50 mm Tris-HCl (pH 7.5), 2 mm EDTA, 2% SDS was added to the sample. The lysed cells were then spotted onto DE81 filters, washed, and air-dried. [3H]Thymidine incorporation into DNA was then measured by liquid scintillation counting. Protein Purification—Gene 2.5 protein-R82C was overproduced and purified using a procedure previously employed to purify WT gene 2.5 protein (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). A 1-liter culture of E. coli BL21(DE3) (Novagen) expressing gene 2.5 protein-R82C was grown, and gene 2.5 protein-R82C was purified by precipitation in polyethyleneimine (pH 7.5), followed by fractionation on an HQ column and a gel filtration column. The protein was 99% pure as determined by denaturing polyacrylamide gel electrophoresis followed by Coomassie Blue staining and was free of contaminating deoxyribonuclease activity (data not shown). Protein concentrations were calculated from UV spectrophotometer readings at 280 mm, using the calculated extinction coefficient at 280 nm of 2.59 × 104 M–1 cm–1 for gene 2.5 protein-R82C (32Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5072) Google Scholar). His-tagged gene 2.5 protein-R82C was purified using a previously described method (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Determining DNA Binding Affinity by Electrophoretic Mobility Shift Assay—The ssDNA binding activity of gene 2.5 protein was assessed by a electrophoretic mobility shift assay (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Gene 2.5 proteins (diluted in a buffer of 20 mm Tris (pH 7.5), 10 mm β-mercaptoethanol, and 500 μg/ml bovine serum albumin) were incubated with 3 nm33P-end-labeled 70-mer oligonucleotide, 15 mm MgCl2, 5 mm dithiothreitol, 50 mm KCl, 10% glycerol, 0.01% bromophenyl blue. ssDNA was separated from ssDNA-protein complex on 10% TBE Ready Gels (Bio-Rad) running in 0.5× Tris/glycine buffer (12.5 mm Tris base, 95 mm glycine, 0.5 mm EDTA). Gels were dried and exposed to a Fujix phosphor imager plate, and the amount of radioactivity was calculated using ImageQuant software. DNA Annealing Assay—The ability of WT gene 2.5 protein to mediate the annealing of homologous strands of DNA was assessed using an in vitro annealing assay developed by Tabor and Richardson (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar). A 310-nucleotide internally labeled ssDNA fragment was generated as described previously (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar, 31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). DNA annealing was assayed in reactions containing 4 nm32P-labeled ssDNA fragment, 20 μm M13 mGP1-2 ssDNA, 20 mm Tris-Cl (pH 7.5), 1 mm dithiothreitol, 10 mm MgCl2, 50 mm NaCl, and various concentrations of gene 2.5 protein. Unless noted otherwise, reactions were incubated at 30 °C for 8 min. Time course experiments were carried out at 30 °C with 10 μm gene 2.5 protein, and the reaction was stopped by the addition of SDS to a final concentration of 0.5%. Reaction products were analyzed on a 0.8% agarose gel at 75 V for 1 h at room temperature, dried under vacuum, and exposed to a Fujix phosphor imager plate, and radioactivity was calculated using ImageQuant software. Plots of the data represent the background-corrected average of three experiments. Electron Microscopy—WT and altered gene 2.5 proteins or E. coli SSB protein were diluted to 500 ng/μl in 20 mm Hepes/NaOH, (pH 7.5), 20% glycerol, mixed with single-stranded WT M13 DNA at 25 ng/μl in a buffer containing 10 mm Hepes/NaOH (pH 7.5), 50 mm NaCl final concentration. MgCl2 was added to the reaction buffer to 10 mm where indicated. Binding reactions with protein/DNA ratios (μg/μg) ranging from 40:1 for WT gene 2.5 protein to 10:1 for mutants and E. coli SSB protein were incubated for 15 min at room temperature in a 50-μl total reaction volume. Following the binding reactions, the samples were fixed with an equal volume of 1.2% glutaraldehyde for 5 min at room temperature and then loaded onto a 2-ml column of Bio-Gel A-5m previously equilibrated in 10 mm Tris·HCl (pH 7.5), 0.5 mm EDTA. The same buffer was then used to elute the sample from the column and 250-μl fractions were collected. Aliquots of the protein-DNA containing fractions were mixed with a buffer containing spermidine (33Griffith J.D. Christiansen G. Annu. Rev. Biophys. Bioeng. 1978; 7: 19-35Crossref PubMed Scopus (181) Google Scholar) for 3 s and quickly applied to a mesh copper grid coated with a thin carbon film, glow-charged shortly before sample application. Following adsorption of the samples to the electron microscopy support for 1–2 min, the grids were subjected to a dehydration procedure in which the water content of the wash solutions was gently replaced by a serial increase in ethanol concentration to 100% and then air-dried. The samples were then rotary shadowcast with tungsten at 10–7 torr and examined in a Philips CM 12 TEM instrument at 40 kV. Micrographs, taken at × 46,000, were scanned using a Nikon LS-4500AF film scanner, and panels were arranged using Adobe Photoshop. Gel Filtration Analysis—Gel filtration analysis was performed as previously described (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Fifty μg of gene 2.5 protein-R82C diluted in buffer S (final concentration 4 μm) were loaded on a Superdex 75 column (Amersham Biosciences). A standard curve of K av versus log M r was generated by applying low molecular weight protein standards (Amersham Biosciences) to the column under the same conditions. Analysis of Protein-Protein Interaction by Surface Plasmon Resonance—The interaction between gene 2.5 protein and T7 DNA polymerase was measured by SPR using the BIACORE 3000 system as described previously (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Briefly, 10 μl of 500 nm histidine-tagged gene 2.5 protein, gene 2.5 protein-R82C, and gene 2.5 protein-Δ26C were immobilized onto separate lanes of a nickel-charged sensor chip NTA (BIAcore). This amount of protein correlated to ∼7,000 resonance units. Ten μl of 500 nm T7 DNA polymerase or bovine serum albumin were passed over the chip, and dissociation of T7 DNA polymerase was monitored for 10 min while passing 100 μl of running buffer over the chip. Each analysis was performed in triplicate and repeated on three separate days. The kinetics of the gene 2.5 protein-T7 DNA polymerase interaction was assessed by binding 50 nm of either WT or mutant histidine-tagged gene 2.5 protein to the nickel-charged chip and then passing 10 μl of 0–50 nm T7 DNA over the chip. BIAevaluation software was used to determine dissociation constants (K D), which were solved using the simultaneous k a/k d data fit. Gene 2.5 Protein-R82C Cannot Support T7 DNA Synthesis or T7 Phage Growth—Gene 2.5 is essential for the growth of bacteriophage T7 (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar). In this study, we examine a mutation, leading to a single amino acid change, arginine 82 to cysteine, that was isolated as part of a screen for lethal mutations in gene 2.5 (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). As such, it was unable to support the growth of T7 phage lacking gene 2.5 (T7Δ2.5) (Table I). Interestingly, this mutation does not affect the function of WT gene 2.5 protein based on the fact that expression of the mutated gene from a plasmid does not inhibit the growth of WT T7 phage (Table I).Table IPlating efficiency of T7 and T7Δ2.5 on E. coli strains containing plasmids expressing WT or mutant T7 gene 2.5 proteinsPlasmidT7T7Δ2.5pETGP2.511pETGP2.5-R82C0.852.0 × 10-5 Open table in a new tab Since gene 2.5 is an essential gene and its product is involved in DNA synthesis in vitro, we examined the ability of gene 2.5 protein-R82C to carry out DNA synthesis in vivo. E. coli cells expressing the WT or mutant gene 2.5 protein were grown to midlog phase and then infected with a T7 phage lacking gene 2.5. At specific time points, aliquots of cells were removed and mixed with radioactively labeled thymidine. After 90 s, the reactions were terminated. Results of such an experiment are shown in Fig. 2. DNA synthesis peaks ∼30 min after infection in cells expressing WT gene 2.5. As a control, no DNA synthesis is observed in cells harboring gene 2.5 lacking the coding sequence for the carboxyl-terminal motif (gene 2.5 protein-Δ26C). Similarly, DNA synthesis declines soon after infection in cells expressing gene 2.5 protein-R82C. Therefore, it is likely that this mutant is lethal because it is defective in some aspect of DNA metabolism. Gene 2.5 Protein-R82C Binds ssDNA—One of the primary attributes of gene 2.5 protein is its ability to bind ssDNA (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). In the current study, we assessed the ability of the altered gene 2.5 proteins to bind ssDNA using an electrophoretic mobility shift assay. Using this method, we previously calculated the dissociation constant (K D) for WT gene 2.5 protein to be 2.6 × 10–6m (31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). As shown in Fig. 3, the mobility of a 70-mer oligonucleotide is retarded as increasing amounts of gene 2.5 protein-R82C are added. Gene 2.5 protein-R82C binds ssDNA with ∼10-fold higher affinity than does the WT protein (K D = 3.0 × 10–7m). Thus, the amino acid alteration causes the protein to bind ssDNA with a higher affinity than WT gene 2.5 protein. Since gene 2.5 protein-R82C retains this vital function, we consider it unlikely that the alteration results in a mis-folded protein. Like other ssDNA binding proteins, WT gene 2.5 protein binds ssDNA with a much higher affinity than double-stranded DNA (2Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). We examined the binding of gene 2.5 protein-R82C to double-stranded DNA using the electrophoretic mobility shift assay. Gene 2.5 protein-R82C bound a double-stranded 70-base pair DNA weakly and in a manner similar to the WT protein (data not shown). Thus, whereas the alteration, arginine 82 to cysteine, conferred higher ssDNA-binding affinity upon gene 2.5 protein, it did not lead to increased double-stranded DNA binding activity. Gene 2.5 Protein-R82C Is Defective in DNA Annealing—Gene 2.5 protein can anneal homologous strands of ssDNA in vitro (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar, 30Hyland E.M. Rezende L.F. Richardson C.C. J. Biol. Chem. 2003; 278: 7247-7256Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 31Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In this study, we looked at the ability of WT and altered gene 2.5 proteins to anneal a 310-nucleotide ssDNA fragment to single-stranded M13 DNA. As previously shown (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar), WT gene 2.5 protein can efficiently anneal these homologous strands of DNA (Fig. 4A). In this reaction, an internally labeled 310-nucleotide ssDNA is mixed with M13 circular ssDNA in the presence of varying concentrations of gene 2.5 protein. The labeled DNA fragment is homologous to a region of the M13 ssDNA. Annealing of the 310-nucleotide fragment to the homologous region of M13 ssDNA does not occur after an 8-min incubation at 30 °C in the absence of gene 2.5 protein (Fig. 4A, lane 1), since we observe a single, rapidly migrating radioactively labeled species on an agarose gel. When the concentration of gene 2.5 protein in the reaction is increased, annealing of the DNA strands begins to occur. In Fig. 4A, lane 4, we observe two species, the faster migrating corresponding to the unannealed 310-nucleotide fragment and a more slowly migrating species corresponding to the annealed product. The more slowly migrating species is present even after extraction with phenol chloroform (data not shown), suggesting that the gel shift is due to the increase in size of the annealed product and not a function of gene 2.5 protein binding to the ssDNA. At even higher concentrations (Fig. 4A), all of the labeled fragment is annealed to the M13 circular ssDNA. As previously shown (16Tabor, S., and Richardson, C. C. (July 9, 1996) U. S. Patent 5,534,407Google Scholar), DNA annealing is not observed under the same conditions when E. coli SSB protein is added to the reaction (Fig. 4B). Instead, a third species that migrates faster than the annealed product and slower than the fragment is observed upon the addition of E. coli SSB protein. Such a gel shift is noted in all protein concentrations tested (Fig. 4B, lanes 2–7). This species migrates more rapidly than the annealed product produced by gene 2.5 protein under the same conditions (Fig. 4B, lane 8). At pH 7.5, DNA annealing by E. coli SSB protein is dependent on the presence of a polyamine (28Christiansen C. Baldwin R.L. J. Mol. Biol. 1977; 115: 441-454Crossref PubMed Scopus (57) Google Scholar). Since we did not include polyamine in our assay, it is not surprising that E. coli SSB protein could not mediate this reaction under the conditions employed in this study. Gene 2.5 protein-R82C is defective in DNA annealing (Fig. 4C). At the highest concentration test (45 μm), only ∼25% of the fragment is converted to annealed product (Fig. 4C, lane 6). Under the same conditions, WT gene 2.5 protein anneals 100% of the fragment at a concentration of 15 μm (Fig. 4A, lane 5). Like E. coli SSB protein, gene 2.5 protein-R82C has a higher affinity for ssDNA than the WT protein. Thus, it is not surprising that we observe the appearance of a band that probably corresponds to a protein-DNA complex as the concentration of gene 2.5 protein-R82C in the reaction is increased (Fig. 4C, lanes 2–6). Next, we compared DNA annealing mediated by the WT protein with annealing mediated by gene 2.5 protein-R82C over a 4-min time period. In Fig. 5, we show that the WT protein anneals nearly all of the labeled fragment in the reaction in less than 3 min. In contrast, when the same concentration of gene 2.5 protein-R82C is added to the reaction, no annealed product is observed over the 4-min time course. Gene 2.5 protein-R82C and E. coli SSB protein both have a higher affinity for ssDNA than WT gene 2.5
Referência(s)