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C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA

2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês

10.1074/jbc.m109.024059

ISSN

1083-351X

Autores

Sharmistha Ghosh, Boriana Marintcheva, Masateru Takahashi, Charles C. Richardson,

Tópico(s)

Bacterial Genetics and Biotechnology

Resumo

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. Single-stranded DNA (ssDNA) 3The abbreviations used are: ssDNAsingle-stranded DNAtrxthioredoxindsDNAdouble-stranded DNATBDthioredoxin binding domainDTTdithiothreitolntnucleotideSSBsingle strand DNA-binding protein. 3The abbreviations used are: ssDNAsingle-stranded DNAtrxthioredoxindsDNAdouble-stranded DNATBDthioredoxin binding domainDTTdithiothreitolntnucleotideSSBsingle strand DNA-binding protein.-binding proteins have been assigned the role of removing secondary structure in DNA and protecting ssDNA from hydrolysis by nucleases (1Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (445) Google Scholar). However, in addition to these mundane roles, ssDNA-binding proteins are now recognized as a key component of the replisome where they physically and functionally interact with other replication proteins and with the primer-template (2Benkovic S.J. Valentine A.M. Salinas F. Annu. Rev. Biochem. 2001; 70: 181-208Crossref PubMed Scopus (272) Google Scholar, 3O'Donnell M. J. Biol. Chem. 2006; 281: 10653-10656Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 4Hamdan S.M. Richardson C.C. Annu. Rev. Biochem. 2009; 78: 205-243Crossref PubMed Scopus (136) Google Scholar). ssDNA-binding proteins are also engaged in DNA recombination and repair (5Lohman T.M. Ferrari M.E. Annu. Rev. Biochem. 1994; 63: 527-570Crossref PubMed Scopus (527) Google Scholar). In view of these multiple roles, it has been difficult to identify the specific defect in genetically altered ssDNA-binding proteins that leads to an observed phenotype. single-stranded DNA thioredoxin double-stranded DNA thioredoxin binding domain dithiothreitol nucleotide single strand DNA-binding protein. single-stranded DNA thioredoxin double-stranded DNA thioredoxin binding domain dithiothreitol nucleotide single strand DNA-binding protein. The crystal structures of several prokaryotic ssDNA-binding proteins have been determined (6Hollis 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 (79) Google Scholar, 7Savvides S.N. Raghunathan S. Fütterer K. Kozlov A.G. Lohman T.M. Waksman G. Protein Sci. 2004; 13: 1942-1947Crossref PubMed Scopus (111) Google Scholar, 8Shamoo Y. Friedman A.M. Parsons M.R. Konigsberg W.H. Steitz T.A. Nature. 1995; 376: 362-366Crossref PubMed Scopus (221) Google Scholar). These proteins have a conserved oligosaccharide-oligonucleotide binding fold (OB-fold) that is thought to bind the ssDNA by means of stacking and electrostatic interactions (6Hollis 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 (79) Google Scholar). Prokaryotic ssDNA-binding proteins also have an acidic C-terminal tail that is essential for bacterial and phage growth (9Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar, 10Burke R.L. Alberts B.M. Hosoda J. J. Biol. Chem. 1980; 255: 11484-11493Abstract Full Text PDF PubMed Google Scholar, 11Hosoda J. Moise H. J. Biol. Chem. 1978; 253: 7547-7558Abstract Full Text PDF PubMed Google Scholar, 12Williams K.R. Konigsberg W. J. Biol. Chem. 1978; 253: 2463-2470Abstract Full Text PDF PubMed Google Scholar, 13Williams K.R. Spicer E.K. LoPresti M.B. Guggenheimer R.A. Chase J.W. J. Biol. Chem. 1983; 258: 3346-3355Abstract Full Text PDF PubMed Google Scholar). The ssDNA-binding protein of bacteriophage T7 is encoded by gene 2.5 (14Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 10173-10177Crossref PubMed Scopus (59) Google Scholar). The gene 2.5 protein (gp2.5) is a homodimer in solution, a structure that is stabilized by its C-terminal tail (9Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar, 15Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar). The C-terminal tail of one monomer of gp2.5 binds in a trans mode to the ssDNA-binding cleft of the other subunit, thus stabilizing the dimer interface observed in the crystal structure (6Hollis 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 (79) Google Scholar). The current model proposes that the positively charged DNA-binding cleft is shielded by the electrostatic charges of the C-terminal tail in the absence of ssDNA, thus facilitating oligomerization of gp2.5. Upon binding ssDNA, the dimer dissociates to allow the C-terminal tail to interact with other replication proteins (16Shokri L. Marintcheva B. Eldib M. Hanke A. Rouzina I. Williams M.C. Nucleic Acids Res. 2008; 36: 5668-5677Crossref PubMed Scopus (23) Google Scholar). The tail modulates the affinity for ssDNA and protein-protein interactions by functioning as a two-way switch (6Hollis 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 (79) Google Scholar, 17Marintcheva B. Marintchev A. Wagner G. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1855-1860Crossref PubMed Scopus (39) Google Scholar). This mode of function is applicable to other prokaryotic ssDNA-binding proteins, namely Escherichia coli SSB protein and T4 gp32 (10Burke R.L. Alberts B.M. Hosoda J. J. Biol. Chem. 1980; 255: 11484-11493Abstract Full Text PDF PubMed Google Scholar, 13Williams K.R. Spicer E.K. LoPresti M.B. Guggenheimer R.A. Chase J.W. J. Biol. Chem. 1983; 258: 3346-3355Abstract Full Text PDF PubMed Google Scholar, 15Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 18Kowalczykowski S.C. Lonberg N. Newport J.W. von Hippel P.H. J. Mol. Biol. 1981; 145: 75-104Crossref PubMed Scopus (258) Google Scholar, 19Newport J.W. Lonberg N. Kowalczykowski S.C. von Hippel P.H. J. Mol. Biol. 1981; 145: 105-121Crossref PubMed Scopus (123) Google Scholar, 20Lonberg N. Kowalczykowski S.C. Paul L.S. von Hippel P.H. J. Mol. Biol. 1981; 145: 123-138Crossref PubMed Scopus (76) Google Scholar, 21Krassa K.B. Green L.S. Gold L. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 4010-4014Crossref PubMed Scopus (38) Google Scholar, 22Nelson S.W. Kumar R. Benkovic S.J. J. Biol. Chem. 2008; 283: 22838-22846Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). gp2.5 is one of four proteins that include the T7 replisome. The other three proteins are the T7 gene 5 DNA polymerase (gp5), its processivity factor, E. coli thioredoxin (trx), and the multifunctional gene 4 helicase-primase (gp4). gp5 and trx bind with high affinity (KD of 5 nm), and the two proteins are normally found in complex (gp5/trx) at a stoichiometry of one to one (23Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). The acidic C-terminal tail of gp2.5 is critical for the interactions of the protein with gp5/trx and gp4 (9Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar, 24Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The C-terminal tail binds to a positively charged segment located in the thumb subdomain of the gp5 (25Hamdan S.M. Marintcheva B. Cook T. Lee S.J. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5096-5101Crossref PubMed Scopus (77) Google Scholar). This fragment, designated the trx binding domain (TBD), is also the site of binding of the processivity factor, E. coli trx, and the C terminus of gp4. The multiple interactions of the C terminus of gp2.5 could thus function to coordinate the dynamic reactions occurring at the replication fork. gp2.5 is known to be critical for establishing coordination during leading and lagging strand DNA synthesis (26Lee 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, 27Hamdan S.M. Loparo J.J. Takahashi M. Richardson C.C. van Oijen A.M. Nature. 2009; 457: 336-339Crossref PubMed Scopus (121) Google Scholar). This C-terminal tail of gp2.5 is an acidic 26-amino acid segment with an aromatic phenylalanine as the C-terminal residue. The C-terminal tail is not seen in the crystal structure because gp2.5Δ26, lacking the tail, was used for crystallization; the wild-type protein did not yield crystals that diffracted (6Hollis 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 (79) Google Scholar). gp2.5ΔF designates a genetically modified gp2.5 lacking the C-terminal phenylalanine. gp2.5ΔF does not support the growth of T7Δ2.5 phage lacking gene 2.5 (28Marintcheva B. Hamdan S.M. Lee S.J. Richardson C.C. J. Biol. Chem. 2006; 281: 25831-25840Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Interestingly, T7 gene 4 protein also has an acidic C-terminal tail with a C-terminal phenylalanine (29Lee S.J. Marintcheva B. Hamdan S.M. Richardson C.C. J. Biol. Chem. 2006; 281: 25841-25849Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Again, the phenylalanine is critical for the interaction of gp4 with gp5/trx (29Lee S.J. Marintcheva B. Hamdan S.M. Richardson C.C. J. Biol. Chem. 2006; 281: 25841-25849Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Further evidence for overlapping binding sites of the C termini of these two proteins comes from studies with chimeric proteins (28Marintcheva B. Hamdan S.M. Lee S.J. Richardson C.C. J. Biol. Chem. 2006; 281: 25831-25840Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 29Lee S.J. Marintcheva B. Hamdan S.M. Richardson C.C. J. Biol. Chem. 2006; 281: 25841-25849Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The C-terminal tails of gp2.5 and gp4 can be exchanged, and the chimeric proteins support the growth of T7 phage lacking the corresponding wild-type protein. We recently designed a screen for suppressors of dominant lethal mutations of gp2.5 (30Marintcheva B. Qimron U. Yu Y. Tabor S. Richardson C.C. Richardson C. Mol. Microbiol. 2009; 72: 869-880Crossref PubMed Scopus (6) Google Scholar). The screen identified mutations in gene 5, the structural gene for T7 DNA polymerase (Fig. 1), which suppresses the lethal phenotype of gp2.5 mutant in which the C-terminal phenylalanine was moved to the penultimate position (gp2.5ΔF232InsF231). One of the altered suppressor genes (gp5, gp5-sup1) encodes a gp5 in which where glycine at position 371 is replaced by lysine (G371K). Whereas the other (gp5-sup2) encodes a protein in which threonine 258 and alanine 411 are replaced by methionine and threonine, respectively (T258M and A411T). The suppressor mutations in gp5 are necessary and sufficient to suppress the lethal phenotype of gp2.5ΔF232InsF231. The affected residues map in proximity to aromatic residues and to residues in close proximity to DNA as seen in the crystal structure of gp5/trx in complex with DNA (31Doublié S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1100) Google Scholar). Throughout this study, gp2.5ΔF232InsF231 mutant will be referred to as gp2.5-FD because it effectively switches the positions of the C-terminal phenylalanine and the adjacent aspartic acid. E. coli SSB protein also has a C-terminal phenylalanine, and recent studies have shown that this residue inserts into a hydrophobic region consisting of exonuclease I of E. coli (45Engler M.J. Lechner R.L. Richardson C.C. J. Biol. Chem. 1983; 258: 11165-11173Abstract Full Text PDF PubMed Google Scholar, 46Lee J. Chastain 2nd, P.D. Griffith J.D. Richardson C.C. J. Mol. Biol. 2002; 316: 19-34Crossref PubMed Scopus (54) Google Scholar). In this study, we have purified the two suppressor DNA polymerases and characterized them individually and in interaction with the other T7 replication proteins. Whereas wild-type gp5 binds with low affinity to gp2.5-FD, the DNA polymerases harboring the suppressor mutations bind with a higher affinity. An interesting finding is that whereas wild-type gp2.5 enables gp5/trx to catalyze strand displacement synthesis at a nick in DNA, gp2.5-FD does not support this reaction. Strand displacement synthesis is necessary for the initiation of leading strand DNA synthesis at a nick because it creates a 5′-single-stranded DNA tail for loading of the T7 helicase (32Lechner R.L. Engler M.J. Richardson C.C. J. Biol. Chem. 1983; 258: 11174-11184Abstract Full Text PDF PubMed Google Scholar). Wild-type gp5, gp5-sup1, and gp5-sup2 plasmids have been described previously in the study identifying gp5 suppressors of the dominant lethal gp2.5-FD (30Marintcheva B. Qimron U. Yu Y. Tabor S. Richardson C.C. Richardson C. Mol. Microbiol. 2009; 72: 869-880Crossref PubMed Scopus (6) Google Scholar). Wild-type gp5, gp5-sup1, and gp5-sup2 were purified from E. coli HMS 174(DE3)/pLysS cells overexpressing their genes as described previously (23Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). The 1:1 complex of polymerase and thioredoxin was purified to apparent homogeneity using three chromatographic steps as follows: phosphocellulose (Whatman), anion exchange Poros HQ (PerSeptive Biosystems), and ceramic hydroxylapatite (Bio-Rad) as described (23Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). Wild-type gp2.5, gp2.5-FD, and gp2.5Δ26 were purified from BL21(DE3)pLysS cells overexpressing their genes as described previously (33Rezende 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). gp4 was purified as described (24Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Surface plasmon resonance analysis was performed using a Biacore 3000 instrument. Wild-type and genetically altered gp2.5 were immobilized (150 response units) on a carboxymethyl-5 chip using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide chemistry. Immobilization was performed in 10 mm sodium acetate, pH 5.0, except for gp2.5Δ26 protein, which was immobilized at pH 4.5 at a flow rate of 10 μl/min. Binding studies were performed in 20 mm HEPES (pH 7.5), 10 mm MgCl2, 250 mm potassium glutamate, 5 mm DTT at a flow rate of 40 μl/min (25Hamdan S.M. Marintcheva B. Cook T. Lee S.J. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5096-5101Crossref PubMed Scopus (77) Google Scholar, 28Marintcheva B. Hamdan S.M. Lee S.J. Richardson C.C. J. Biol. Chem. 2006; 281: 25831-25840Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The chip surface was regenerated using 1 m NaCl at a flow rate of 100 μl/min. As a control, a flow cell was activated and blocked in the absence of protein to account for changes in the bulk refractive index. Apparent binding constants were calculated under steady-state conditions, and the data were fitted using BIAEVAL 3.0.2 software (Biacore). DNA polymerase activity was measured using M13 ssDNA as a template as described previously (23Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). The reaction contained 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, 20 nm M13 mGP1–2 ssDNA annealed to a 24-nt oligonucleotide, with 500 μm each of dATP, dCTP, dGTP, and [3H]dTTP (2 cpm/pmol), 50 μg/ml bovine serum albumin, and 0.3 nm gp5/trx in a total volume of 10 μl. Reaction mixtures were incubated at 37 °C for the indicated times and stopped by addition of 5 μl of 0.25 m EDTA (pH 7.5). The incorporation of [3H]dTMP was measured on DE81 filter disks as described (23Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). The 3′–5′-exonuclease activity of T7 DNA polymerase was measured using uniformly labeled M13 [3H]dsDNA as described previously (34Kumar J.K. Tabor S. Richardson C.C. J. Biol. Chem. 2001; 276: 34905-34912Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The M13 [3H]dsDNA was prepared by annealing the 24-nt oligonucleotide to M13 mGP1–2 DNA and then extending the primer by 200 nm T7 DNA polymerase in a 50-μl reaction mixture containing 40 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 10 mm DTT, 50 mm potassium glutamate, 0.5 mm each of dATP, dCTP, dGTP, and [3H]dTTP (3000 Ci/mmol). After incubation at 37 °C for 10 min, the DNA was extracted with phenol/chloroform and then purified by passing through Biospin 6 columns (Bio-Rad) to remove free nucleotides. M13 [3H]ssDNA was prepared by alkali denaturation of 3H-labeled M13 dsDNA (34Kumar J.K. Tabor S. Richardson C.C. J. Biol. Chem. 2001; 276: 34905-34912Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). M13 [3H]dsDNA was treated by 50 mm NaOH at 37 °C for 15 min followed by neutralization with HCl. Reaction mixtures (10 μl) contained 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, 0.5 nmol (in terms of total nucleotides) of 3H-labeled M13 mGP1–2 dsDNA, and 0–20 nm gp5/trx. After incubation for 20 min, the reaction was stopped by the addition of EDTA at a final concentration of 125 mm. The hydrolysis of DNA was measured by spotting the reaction mixture on DE81 filters and by determining the amount of radioactivity remaining in DNA by scintillation counting. Leading strand DNA synthesis catalyzed by gp5/trx and gene 4 helicase was measured using circular M13 containing a preformed replication fork (35Delagoutte E. von Hippel P.H. Biochemistry. 2001; 40: 4459-4477Crossref PubMed Scopus (60) Google Scholar). The replication fork (see Fig. 4A, inset) was constructed by annealing M13 mGP1–2 ssDNA to an oligonucleotide (5′-TAATTCGTAATCATCATGGTCATAGCTGTTTCCT-3′). The oligoribonucleotide was then extended by gp5/trx to obtain double-stranded DNA. Strand displacement DNA synthesis was carried out in a reaction mixture (10 μl) containing 10 nm circular M13 DNA containing a preformed replication fork, 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 5 mm DTT, 50 mm potassium glutamate, 500 μm each of dCTP, dGTP, and dTTP, 0.05 μCi of [α-32P]dATP, 10 nm gp4 (hexamer), and 2.5–20 nm of wild-type or variants of gp5/trx. gp5/trx and gp4 were incubated on ice for 15 min, and reactions were initiated by transferring to 37 °C. After 10 min, the reaction was stopped by the addition of EDTA to a final concentration of 125 mm. Where indicated, gp2.5, gp2.5-FD, and gp2.5Δ26 protein were present at 4 μm unless otherwise specified. Leading strand synthesis on nicked DNA was carried out using a circular M13 double-stranded DNA containing a nick (see Fig. 4A, inset). The DNA was prepared by annealing a 24-nt oligonucleotide to M13 mGP1–2 ssDNA. The primer was extended to make a fully circular dsDNA in a reaction containing 10 nm gp5/trx, 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, with 500 μm each of dATP, dCTP, dGTP, and dTTP, 50 μg/ml bovine serum albumin. The reaction was incubated at 37 °C for 3 min and stopped by heating at 75 °C for 5 min. The nicked DNA was extracted with phenol/chloroform and then purified by passing through Biospin 6 columns (Bio-Rad) to remove free nucleotides. The structure of the nicked DNA was confirmed by analysis on a denaturing agarose gel. Nicked DNA was also prepared by incubating supercoiled pBR322 DNA with Nb.Bsml nicking enzyme (New England Biolabs) as described by the manufacturer. The enzyme was inactivated by heating the reaction to 80 °C for 20 min (as specified by New England Biolabs). The conversion of the supercoiled DNA to a relaxed form was confirmed by gel analysis. Leading strand synthesis reactions were the same as described above. To examine the role of gp2.5 proteins in this reaction, varying amounts of gp2.5, gp2.5-FD, and gp2.5Δ26 protein were added to the reaction. DNA synthesis was monitored by the amount of [α-32P]dAMP incorporated into DNA (23Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). To visualize the products of DNA synthesis, the DNA products were denatured and analyzed by electrophoresis in a 0.6% alkaline agarose gel. Coordinated DNA synthesis reactions were carried out using a mini-circle substrate as described previously (26Lee 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). The nucleotide sequence of the mini-circle DNA was modified slightly to more easily differentiate synthesis of the leading strand from that on the lagging strand using [α-32P]dTTP and [α-32P]dATP, respectively. The sequences used in constructing the mini-circle are as follows: 5′-CCA CCC CAA AAA CAC CAA CAA CCC AAC ACC ACA CAA CAC ACC ACA AAA CCA CAC GAC CAA AAC CAC CAC C-3′ for the 70-base circle and 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T GGT GGT GGT TTT GGT CGT GTG GTT TTG TGG TGT GTT GTG TGG TGT TGG GTT GTT GGT GTT TTT GGG GTG G-3′ for the complementary strand to the mini-circle, which will result in a 5′ overhang. Preparation of the mini-circle is identical to that described previously (26Lee 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, 36Johnson D.E. Takahashi M. Hamdan S.M. Lee S.J. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5312-5317Crossref PubMed Scopus (39) Google Scholar). The replication reaction is carried out for 5 min at 30 °C, with a mini-circle concentration of 32 nm, 28 nm gp5/trx, 4 nm gp4, 2 μm gp2.5, 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, with 500 μm each of dATP, dCTP, dGTP, and dTTP, and 50 μg/ml bovine serum albumin. The rolling-circle substrate is designed in a way that leading strand synthesis is monitored by incorporation [32P]dTMP and lagging strand synthesis by [32P]dAMP. Radioactive products were separated and visualized on a 0.8% alkaline agarose gel. Aside from removing secondary structures upon binding ssDNA, gp2.5 also coordinates protein-protein and protein-DNA interactions at the replication fork (1Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (445) Google Scholar, 15Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 25Hamdan S.M. Marintcheva B. Cook T. Lee S.J. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5096-5101Crossref PubMed Scopus (77) Google Scholar, 26Lee 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, 27Hamdan S.M. Loparo J.J. Takahashi M. Richardson C.C. van Oijen A.M. Nature. 2009; 457: 336-339Crossref PubMed Scopus (121) Google Scholar). For example, gp2.5 physically interacts with both T7 DNA polymerase in complex with thioredoxin (gp5/trx) and the gene 4 helicase-primase (25Hamdan S.M. Marintcheva B. Cook T. Lee S.J. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5096-5101Crossref PubMed Scopus (77) Google Scholar, 28Marintcheva B. Hamdan S.M. Lee S.J. Richardson C.C. J. Biol. Chem. 2006; 281: 25831-25840Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The acidic C-terminal tails of gp2.5 and gp4 both bind to two basic loops in the TBD of gp5 (25Hamdan S.M. Marintcheva B. Cook T. Lee S.J. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5096-5101Crossref PubMed Scopus (77) Google Scholar). In the course of examining this electrostatic interaction, the important role of the C-terminal phenylalanine of gp2.5 emerged (28Marintcheva B. Hamdan S.M. Lee S.J. Richardson C.C. J. Biol. Chem. 2006; 281: 25831-25840Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This aromatic residue, as well as the overall negative charge of the C terminus, is essential for its interaction with gp5/trx. gp2.5 lacking the C-terminal phenylalanine cannot support the growth of T7 phage lacking gene 2.5 (28Marintcheva B. Hamdan S.M. Lee S.J. Richardson C.C. J. Biol. Chem. 2006; 281: 25831-25840Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This lethal phenotype is also seen with gp2.5-FD, in which the C-terminal phenylalanine (Phe-232) has been switched with the adjacent aspartic acid (Asp-231). This latter phenotype enabled the identification of suppressor mutations in gene 5 of bacteriophage T7 that resulted in changes in residues that may engage DNA in the DNA binding crevice of the polymerase (Fig. 1). These altered polymerases, designated gp5-sup1 and gp5-sup-2, both support the growth of T7Δ5 phage, lacking gene 5, and both can support the growth of T7Δ2.5 phage complemented with gp2.5-FD lacking the C-terminal phenylalanine (28Marintcheva B. Hamdan S.M. Lee S.J. Richardson C.C. J. Biol. Chem. 2006; 281: 25831-25840Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Both suppressor polymerases support the growth of T7Δ5 phage expressing wild-type gp2.5. We have purified these two suppressor DNA polymerases and examined their biochemical properties and interactions with other T7 replication proteins. gp5 expressed in wild-type E. coli exists in a one to one complex (gp5/trx) with its processivity factor trx (37Mark D.F. Richardson C.C. Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 780-784Crossref PubMed Scopus (176) Google Scholar). In all the studies described here, the polymerases are in complex with trx and are designated as gp5/trx, gp5-sup1/trx, and gp5-sup2/trx. The polymerization of nucleotides catalyzed by gp5-sup1/trx and gp5-sup2/trx was compared with that catalyzed by wild-type gp5/trx (Fig. 2A). The initial rates of DNA synthesis with purified enzymes were measured following the incorporation of [3H]dTMP into DNA using primed M13 ssDNA. The rate of DNA synthesis of gp5-sup1/trx and gp5-sup2/trx is similar to that of wild-type gp5/trx. The steady-state rate constants for polymerization (kpol) for gp5/trx, gp5-sup1/trx, and gp5-sup2/trx are 71, 82, and 75 s−1, respectively. Taken together, we conclude that the replacement of Gly → Lys at position 371 in gp5-sup1 or the simultaneous substitution of Thr → Met and Ala → Thr at positions 258 and 411, respectively, in gp5-sup2 does not significantly affect their polymerization activity. T7 DNA polymerase, like other members of the polymerase I family of DNA polymerases, has an N-terminal exonuclease domain (31Doublié S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature.

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