Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10173-10177, November 1993 Biochemistry T7 2.5 protein: An essential protein for DNA replication (DNA binding protein/recombination/T7 DNA polymerase) YOUNG TAE KIM* AND CHARLES C. RICHARDSON Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Contributed by Charles C. Richardson, July 22, 1993

ABSTRACT The product of gene 2.5 of bacteriophage T7, 15). Both direct and indirect evidence support an interaction a single-stranded DNA binding protein, physically interacts with between these essential replication proteins and the T7 gene the phage-encoded gene S protein (DNA polymerase) and gene 2.5 protein. 4 proteins ( and ) and stimulates their activities. T7 gene 2.5 protein stimulates DNA synthesis catalyzed by Genetic analysis ofT7 phage defective in gene 2.5 shows that the the T7 DNA polymerase/ complex on single- gene 2.5 protein is essential for T7 DNA replication and growth. stranded DNA templates (4, 5, 11, 14) and increases the T7 phages thatcontain null mutants ofgene2.5 were constructed processivity ofthe reaction (11). It has been shown by affinity by . These gene 2.5 null mutants chromatography and fluorescence emission anisotropy that contain either a deletion ofgene2.5 (T7A2.5) or an insertion into T7 DNA polymerase and gene 2.5 protein physically interact gene 2.5 and cannot grow in (efficiency of with a dissociation constant of 1.1 ,uM (11). Similarly, inter- plating, <10-8). After infection of E. coli with T7A2.5, host action of the T7 helicase/primase with gene 2.5 protein has DNA synthesis is shut off, and phage DNA synthesis is reduced been inferred from the ability ofgene 2.5 protein to stimulate to <1% ofphage DNA synthesis in wild-type T7-infected E. coli synthesis of primers (10, 16). The T7 helicase/primase-gene cells as measured by incorporation of [3H]thymidine. In con- 2.5 protein interaction has also been confirmed by affinity trast, RNA synthesis is essentially normal in T7A2.5-infected chromatography (11). We have recently found (unpublished cells. The defects in growth and DNA replication are overcome data) that the C-terminal acidic domain of gene 2.5 protein is by wild-type gene 2.5 protein expressed from a plasmid har- required for T7 growth in vivo and it participates in gene 2.5 boring the T7 gene 2.5. dimerization and protein-protein interactions. The role ofgene 2.5 protein in recombination is not as well Single-stranded DNA binding proteins (SSBs), such as Esch- understood. Sadowski et al. (17) demonstrated that extracts erichia coli SSB and T4 gene 32 protein, are essential of T7 phage-infected cells contain an activity that promotes components of DNA metabolism in prokaryotic cells (1-3). renaturation of complementary single strands and suggested The gene 2.5 protein of bacteriophage T7, originally isolated that this activity resided in the T7 SSB. Recent studies (S. based on its strong affinity for single-stranded DNA and its Tabor and C.C.R., unpublished results) have, in fact, dem- stimulate DNA T7 DNA onstrated that the gene 2.5 protein facilitates renaturation of ability to synthesis by polymerase homologous single-stranded DNA even more efficiently than (4, 5), is thought to be analogous to these well characterized does E. coli recA protein, E. coli SSB, or T4 gene 32 protein. SSBs. Like E. coli SSB and T4 gene 32 protein, gene 2.5 A determination of the definitive role of the T7 gene 2.5 protein has been implicated in T7 DNA replication, recom- protein in DNA replication and recombination in vivo is bination, and repair (6-11). We purified gene 2.5 protein from dependent on genetic analysis of gene 2.5 mutants. Previ- cells overexpressing the gene and characterized its physical ously, T7 phage containing mutations in gene 2.5 have been properties and interactions with DNA (6). Gene 2.5 protein isolated based on their inability to grow on E. coli strains that exists as a dimer of two identical subunits of Mr 25,562. It have a defective SSB (8). These T7 mutant phages contain an binds specifically to single-stranded DNA with a stoichiom- amber mutation in gene 2.5 that leads to synthesis of a etry of -7 nt bound per monomer of gene 2.5 protein and shortened polypeptide -90% of the length of the wild-type extends the length of the DNA molecules as measured by protein (9). These gene 2.5 mutant phages can grow on electron microscopy. The binding constant of gene 2.5 pro- wild-type E. coli strains but not on strains expressing a tein for single-stranded DNA is -2.5 x 106 M-1, as deter- temperature-sensitive SSB at the nonpermissive tempera- mined by fluorescence quenching and nitrocellulose filter ture. Furthermore, these T7 phages are defective in recom- binding assays (6). Fluorescence studies suggest that tyrosine bination (8). Recently, however, a mutational analysis has residue(s) on gene 2.5 protein interacts with single-stranded identified T7 gene 2.5 mutants that cannot grow even in E. DNA, whereas tryptophan residues do not (6). coli strains producing wild-type SSB (F. W. Studier, per- In T7 DNA replication, the gene 2.5 protein has the sonal communication). We show that T7 phages with a potential to play one of several essential roles. At the T7 deletion ofgene 2.5 (T7A2.5) do not grow in wild-type E. coli replication fork, three proteins, the products of T7 4 and have no detectable T7 DNA replication; the T7A2.5 and 5 and the host trxA gene, account for the fundamental phages grow normally in E. coli strains expressing wild-type reactions (12, 13). Gene 5 protein is a DNA polymerase, gene 2.5 from a plasmid. catalyzing the polymerization of nucleotides with low pro- cessivity (14, 15). E. coli thioredoxin, the product ofthe trxA MATERIALS AND METHODS gene, binds to gene S protein in a 1:1 stoichiometry and Bacterial Strains. E. coli HMS157 (F- recB21 recC22 confers processivity on the polymerization reaction by in- sbcA5 endA gal thi sup) (laboratory collection), E. coli creasing the affinity ofthe enzyme for a primer/template (14, Abbreviations: SSB, single-stranded DNA binding protein; moi, The publication costs of this article were defrayed in part by page charge multiplicity of infection. payment. This article must therefore be hereby marked "advertisement" *Present address: Department of Microbiology, National Fisheries in accordance with 18 U.S.C. §1734 solely to indicate this fact. University of Pusan, Pusan, 608-737, Korea. 10173 Downloaded by guest on September 29, 2021 10174 Biochemistry: Kim and Richardson Proc. Natl. Acad. Sci. USA 90 (1993) HMS174 (F- hsdR rK,2- mKj2+ recAl) (18), E. coli HB101 using two primers with an EcoRI site within both primers [F- A(mcrCmrr) leu supE44 aral4 galK2 lacYl proA2 (5'-CGACGAATTCTAAGTGGAACTGCGGG-3' and 5'- rpsL20(Strr) xyl-5 mtl-l recA13] (19), E. coli HMS262 (F- CGACGAATTCCCTTTAGCGCCGTAAC-3'). The PCR- hsdR pro leu-lac-thi-supE tonA-trxA) (laboratory collec- generated fragment of T7 gene 2 was cloned into BamHI- tion), E. coli JH21(F- pcnB80, AtrxA307) (20), and E. coli linearized pBR322 to generate pGP2. pGP2 was digested by AN1(F- AtrxA307, metE::TnlO), a derivative ofE. coli C600 EcoRI, and the PCR-generated gene 2.8 fragment was cloned (21) with P1 transduction (22) have been described. Growth into the EcoRI site of pGP2. The resulting plasmid is pGP2- and manipulation of bacteriophage T7 and E. coli were 2.8. Finally, the trxA DNA was cloned into performed as described (23, 24). PCR-produced Construction of Gene 2.5 Plasmids. DNA fragments were pGP2-2.8 that had been digested with Sma I at the junction prepared and cloned by standard procedures (25). E. coli ofgenes 2 and 2.8. The isolation ofa plasmid containing gene HB101 was transformed with the designated plasmids (26). 2-trxA-gene 2.8 was determined by PCR with suitable prim- Three different plasmids containing a wild-type gene 2.5 were ers. The resulting plasmid, pGP2T2.8, expresses a functional constructed by a standard PCR protocol (27). To construct thioredoxin. pGP2.5::trxA and pGP2T2.8 were transformed the first plasmid, two primers, a 5'-end primer with an Nde toE. coli HMS157, generating E. coli HMS157/pGP2.5::trxA I site (5'-CGTAGGATCCATATGGCTAAGAAGATTT- and E. coli HMS157/pGP2T2.8, respectively. E. coli TCACCTC-3') and a 3'-end primer with a BamHI site (5'- HMS157/pGP2T2.8 was grown in LB medium at 37°C with CGTAGGATCCACTTAGAAGTCTCCGTC-3'), were used vigorous shaking to A600 = 0.5 and infected [multiplicity of to amplify T7 DNA sequences 9157-9862 containing gene 2.5 infection (moi) = 0.1] with wild-type T7 phage. After cell coding sequence (28). PCR-generated DNA fragments were lysis, phages were harvested as described (31). To select and incubated with the large fragment ofE. coli DNA polymerase amplify the recombinant (T7A2.5::trxA), HMS262 cells con- I in the presence of the four nucleoside 5'-triphosphates, and taining pGP2.5-1 that supplements wild-type gene 2.5 protein the resulting fragments containing blunt ends were then were grown to midexponential phase and infected with the ligated into the EcoRV site of plasmid pBR322 (29). The lysate produced from wild-type T7 infection of E. coli resulting plasmid pGP2.5-1 contains the gene 2.5 protein HMS157/pGP2T2.8. After cell lysis, amplified T7A2.5::trxA coding sequence under control of the pBR322 tetracycline was plated onE. coli HMS262/pGP2.5-1, and the presence of gene promoter. the trxA gene and the absence of gene 2.5 in the T7 The second plasmid contains gene 2.5 under the control of were confirmed by PCR and restriction analysis ofindividual a T7 RNA polymerase promoter. The 700-bp Nde I/BamHI T7 plaques. The resulting T7 phage (T7A2.5::trxA) substi- fragment containing gene 2.5 was excised from pGP2.5-1 and tutes trxA for gene 2.5. Isolation of T72.5::trxA phage was then cloned into the Nde I and BamHI sites ofplasmid pT7-7, carried out by an analogous procedure with plasmid generating plasmid pGP2.5-2. Plasmid pT7-7, constructed by pGP2.5::trxA instead of pGP2T2.8. S. Tabor (Harvard Medical School, Boston), contains the T7 Measurements of DNA and RNA Synthesis. DNA and RNA RNA polymerase promoter 410 as well as a strong translation synthesis was measured essentially as described (32). E. coli initiation region prior to the polylinker (30). HMS174 cells were grown with shaking at 30°C in M9 CAA The third plasmid, pGP2.5-3, contains the T7 sequence of medium. At 3 x 108 cells per ml, the bacteria were infected the C-terminal half ofgene 2, the entire sequence ofgene 2.5, with the indicated T7 phage at moi = 7. At the indicated and all of gene 2.8 (T7 sequence at nt 8961-10279). Plasmid times, aliquots (0.2 ml) ofphage-infected cells were removed pGP2.5-3 was constructed as follows. Using the two primers and placed in tubes containing 10 ,ul of [3H]thymidine (50 5'-CGTAGGATTCTCGGAGCATTCC-3' and 5'-CGTAG- ,uCi/ml; 1 Ci = 37 GBq) or [3H]uridine (50 ,uCi/ml) to GATCCACTTAGAAGTCTCCGTC-3', a PCR-generated measure DNA or RNA synthesis, respectively. After 90 sec fragment was created and inserted by blunt-end ligation into of incubation at 30°C, growth was terminated by addition of plasmid pBR322 that had been linearized by BamHI digestion 3 ml of cold 5% trichloroacetic acid. The acid-insoluble and followed by filing-in using the large fragment of E. coli material was collected on GF/C filters (Whatman) and DNA polymerase I. The resulting pGP2.5-3 clone was se- washed three times with 3 ml of ice-cold 1 M HCl and two lected and shown to contain an insert of the correct size and times with 3 ml of 95% ethanol. The acid-insoluble radioac- orientation. All the clones generated from PCR-amplified was measured a DNA were sequenced and found to be free of mutations. tivity in toluene-based solvent in a liquid Construction of Gene 2.5 Mutants of Bacteriophage T7. scintillation counter. Gene 2.5 mutant phages were constructed by homologous In Vivo Labeling of T7 DNA. E. coli HMS174 cells were recombination (18). To construct a gene 2.5 null mutant, we grown to 3 x 108 cells per ml in M9 CAA medium and infected used two different plasmids as in vitro recombination sub- with various T7 phage at moi = 3-5. At the desired times after strates. The first plasmid, pGP2.5::trxA, inserts the E. coli infection, aliquots (2 ml) were removed and [3H]thymidine thioredoxin gene (trxA) into the middle of gene 2.5. The (80 Ci/mmol) was added to the infected cells (final concen- second plasmid, pGP2.5T2.8, replaces gene 2.5 ofT7 with the tration, 40 Ci/ml). Labeling was stopped by addition of an E. coli thioredoxin gene (trxA). Plasmid pGP2.5::trxA was equal volume offreshly made stop solution (75% ethanol/2% constructed as follows. E. coli trxA was amplified by PCR phenol/21 mM CH3COONa, pH 5/2 mM EDTA). The ra- with two primers, each containing an Mlu I site within its dioactively labeled T7 DNA was isolated as described (33), sequence (5'-CAACACGCGTGGCTTATTCCTGTG-3' and cleaved with Xmn I, and electrophoresed through 0.8% 5'-CGAACGCGTTTAAGCCAGGTTAG-3'). PCR-ampli- agarose gel in TBE (12.1 g ofTris base per liter/4.1 g ofboric fled DNA fragments of trxA were cloned into pGP2.5, which acid per liter/0.74 g ofNa2EDTA per liter) at =0.5 V/cm. The had been made linear by cleavage with Mlu I, generating gels were treated with EN3HANCE (NEN), dried, and pGP2.5::trxA. The resulting plasmid expresses a truncated exposed to XAR Kodak film with an intensifying screen at gene 2.5 protein but an active thioredoxin. Plasmid pGP2T2.8 -70°C; bands were identified by autoradiography. was constructed as follows. Part of gene 2, 8961-9096 in the Other Methods. Plating efficiencies of T7 wild-type and nucleotide sequence of T7 DNA, was amplified by using two T7A2.5::trxA phage were measured as follows. Bacterial primers containing a BamHI site within both primers (5'- strains were grown to 2 x 108 cells per ml. Various T7 phages CGTAGGATCCTCGGAGCATTCC-3' and 5'-GAAT- in LB were diluted (0.1 ml) and mixed with 0.2 ml ofbacterial TCGTCGACGGATCCCGGGCGTATTACTTCGGTGCT- culture and 3 ml of top agar, plated on LB or LB/ampicillin 3'). Similarly, gene 2.8 (nt 9854-10279) was amplified by plates, and allowed to incubate at 30°C. Downloaded by guest on September 29, 2021 Biochemistry: Kim and Richardson Proc. Natl. Acad. Sci. USA 90 (1993) 10175 RESULTS Table 1. Plating efficiencies of T7 phages on various E. coli strains Construction of Bacteriophage T7 Gene 2.5 Mutants and Gene 2.5 Expression Vectors. T7 phage defective in gene 2.5 Efficiency of plating were constructed by homologous recombination. As shown Strain/plasmid 17 (WT) T7A2.5::trxA T72.5::trxA in Fig. 1, the plasmid pGP2T2.8 provides the flanking se- HMS262 trxA- 0 <10-9 <10-9 quences (gene 2 and gene 2.8) of gene 2.5 for genetic HMS262/pGP2.5-1 <10-9 0.95 0.92 crossover but with the replacement ofgene 2.5 by the E. coli JH21/pGP2.5-1 <10-9 0.93 0.95 thioredoxin gene trxA. Recombinant T7 phage having gene AN1/pGP2.5-1 <10-9 0.94 0.92 2.5 replaced by trxA (T7A2.5::trxA) by the resulting cross- <10-9 over were selected for growth on an E. coli trxA- strain (E. AN1/pGP2.5-2* 0.91 0.93 coli HMS262) in which wild-type gene 2.5 protein is provided AN1/pGP2.5-3* <10-9 0.93 0.95 from one ofthe three plasmids containing gene 2.5 (pGP2.5-1, HMS157 1 <10-8t 10-2 pGP2.5-2, pGP2.5-3). pGP2.5-1 and pGP2.5-2 contain gene Efficiency of plating was calculated by dividing the number of 2.5 under the control of the tetracycline gene promoter of plaque-forming units on a given strain by the number of wild-type pBR322 and the T7 RNA (WT) 17 plaque-forming units on E. coli HMS157. 17A2.5::trxA and polymerase promoter of pT7-7, 172.5::trxA phages were selected and propagated on E. coli respectively. pGP2.5-3 contains gene 2.5 flanked by the HMS262/pGP2.5-1 cells. proximal half of gene 2 on one side and by all of gene 2.8 on *E. coli HMS262/pGP2.5-2, JH21/pGP2.5-2, HMS262/pGP2.5-3, the other; expression of gene 2.5 is under the control of its and JH21/pGP2.5-3 cells show similar values ofefficiency ofplating own promoter. T7A2.5::trxA lacks the DNA sequence at nt of 17A2.5::trxA and 172.5::trxA phages. 9097-9853 on the T7 DNA molecule (28). This region contains tWhen T7A2.5::trxA was selected and propagated on E. coli the entire gene 2.5 sequence as well as the promoter for gene HMS262/pGP2.5-2 and HMS262/pGP2.5-3 cells, wild-type 17 2.5. A second T7 gene 2.5 mutant, T72.5::trxA, prepared phages arose at frequencies of 10-5 and 10-2, respectively. similarly to T7A2.5::trxA, has the E. coli trxA gene inserted into the coding sequence of gene 2.5. on E. coli HMS262/pGP2.5-1 cannot grow in E. coli HMS262 When T7A2.5::trxA phages are grown in E. coli HMS262 trxA- and E. coli HMS157; the plating efficiencies are <10-9 containing either plasmid pGP2.5-2 or pGP2.5-3, wild-type and <10-8, respectively. Similar results were obtained when T7 phage appears in the resulting lysate at a frequency of 10o- T7A2.5: :trxA phage was plated on other trxA mutant or 10-2, respectively (Table 1). Presumably, these wild-type strains-E. coli JH21 and AN1 (data not shown). T7 phage T7 phages are produced by recombination between with an insertion of the E. coli thioredoxin gene in gene 2.5 T7A2.5::trxA and the complementing plasmid (pGP2.5-2 or (T72.5::trxA) also cannot grow on these strains, although pGP2.5-3). However, when complementation is provided by wild-type T7 revertants arise at a frequency of -2% (titered pGP2.5-1, no wild-type T7 revertants (<10-8) are detected on E. coli HMS157) as a result ofhomologous recombination since no sequence homology exists through the flanking during propagation (Table 1). In control experiments, both regions ofgene 2.5 or the promoter regions ofT7. In the case gene 2.5 mutant T7 phages grow normally on E. coli strains of T72.5::trxA, wild-type revertants arise at a frequency of expressing gene 2.5 from a plasmid harboring the cloned gene %2% via homologous recombination during propagation on (Table 1). The results show that gene 2.5 is essential for the E. coli strains used for complementation. growth of T7. Gene 2.5 Is Essential for T7 Growth. Biochemical evidence DNA and RNA Synthesis in T7A2.5-Infected Cells. In an suggests that the gene 2.5 protein plays crucial roles in attempt to define the biochemical lesions responsible for the several aspects of T7 DNA metabolism. Consequently, we inability of T7 gene 2.5 mutants to grow in E. coli cells, we have used the T7 gene 2.5 mutants T7A2.5::trxA and examined DNA and RNA synthesis in E. coli cells infected T72.5: :trxA to examine the role ofgene 2.5 protein in vivo. As with the mutant phage. The kinetics of DNA synthesis after shown in Table 1, T7A2.5::trxA phage selected and amplified infection of E. coli HMS174 with T7A2.5::trxA phage were compared (Fig. 2) with those obtained with wild-type phage under identical conditions. In wild-type T7-infected cells the rate of DNA synthesis increased rapidly %7 min after infec- tion and reached a maximum rate 20 min after infection, after pGP2T2.8 ori which the cells begin to lyse. In striking contrast, there is a decrease in DNA synthesis after infection with 17A2.5::trxA, BamHI EcoRI presumably due to the shut-off of host DNA synthesis. At 20 min after infection, a time when wild-type T7 DNA replica- tion is maximal, there is no detectable DNA synthesis in the T7WT H ~~~Gene 2 Gene 2. 8 mutant-infected cells (Fig. 2). The defect in DNA synthesis T7WT [ Gene Gee. ee. 60 I Select A2.5::tx,4 T7 WT

T7A2.5::trxA / T 7A2.5::trxA [::1 1 } 40 pGP2.5 (WTl O I \ X FIG. 2. Time course of DNA FIG. 1. Construction of T7A&2.5::trxA. Plasmid pGP2T2.8 is a synthesis in wild-type (WT) 17- derivative of pBR322 containing 17 gene 2-E. coli thioredoxin gene and cells. Rates (trxA)-T7 gene 2.8 sequences between the BamHI and EcoRI sites. c&. ] 2\ 17A&2.5-infected 2020 of DNA synthesis were measured DNA fragments of gene 2, gene 2.8, and the E. coli trxA gene were l / / bas described at various intervals amplified by PCR, and pGP2T2.8 was constructed as described. after infection of E. coli HMS174 During wild-type T7 infection of HMS157/pGP2T2.8, pGP2T2.8 cells with T7 wild-type (o) or recombined homologously with 17 wild-type (WT) phage to generate 1T7A2.5::trx4 1)>>T7A2.5::trxA (o) phages and after the T7A2.5::trxA phage. The presence of the trxA gene and the infection of E. coli HMS174/ absence of gene 2.5 in 17A2.5::trxA phage was confirmed by PCR 0 20 40 pGP2.5-lwithT7A2.5::trxAphage and restriction analysis. TimeAfterInfection (min.) (v) at a moi of 7 at 30°C. Downloaded by guest on September 29, 2021 10176 Biochemistry: Kim and Richardson Proc. Natl. Acad Sci. USA 90 (1993)

T7A2.5/ after infection with T7 wild-type phage under identical con- T7 WT T7A2.5 pGP2.5WT ditions (Fig. 4). Min.: 5 10 15 5 10 15 5 10 15 1 DISCUSSION J:~2 We have used a genetic approach to examine the role of the - I.. -J gene 2.5 protein of bacteriophage T7. T7 lacking gene 2.5 - 5 -6 does not grow in E. coli and is defective in DNA synthesis. - Both defects are overcome by gene 2.5 protein produced 7 from a plasmid containing the cloned gene 2.5. Earlier studies _8 had suggested that E. coli SSB could substitute for the T7 -9 gene 2.5 protein in vivo (8). However, the gene 2.5 mutant -10 used in those studies produced a shortened polypeptide that contained a significant portion of the intact gene 2.5 protein - 12 (9). Our results show clearly that E. coli SSB cannot substi- 1 2 3 4 5 6 7 8 9 tute for gene 2.5 protein in vivo. The multiple roles ofSSBs in DNA metabolism have made FIG. 3. Replication of wild-type 17 and T7A2.5 phage DNA. E. it difficult to define precisely their essential nature in vivo. In coli HMS174 cells were infected with wild-type T7 phage (T7 WT) or E. coli, for example, SSB plays important roles in DNA T7A2.5::trxA (M7A2.5). E. coli HMS174/pGP2.5-1 cells were in- replication as well as in DNA recombination and repair (1-3, fected with T7A2.5::trxA phage [T7A2.5/pGP2.5(WT)]. At 5 (lanes 1, 34). Strains carrying mutations in the structural gene for SSB, 4, and 7), 10 (lanes 2, 5, and 8), and 15 (lanes 3, 6, and 9) min ssb, are temperature sensitive for growth and DNA synthesis postinfection, cells were labeled with [3H]thymidine for 35 sec. The (35) and are defective in DNA repair and recombination (36). labeled 17 DNA was isolated, digested with Xmn I, and treated as described. Restriction fragments are numbered 1-12 and the con- T7 gene 2.5 protein and E. coli SSB have extensive sequence catameric joint fragment is labeled J. homology; 24% of the amino acids are identical and another 47% represent conserved changes (37). In bacteriophage T4, in the T7 gene 2.5 mutant-infected cells could be overcome by the roles ofgene 32 protein have also been well characterized complementation with gene 2.5 protein provided from a both in vitro and in vivo. Genetic studies have demonstrated plasmid (Fig. 2). However, the pattern of DNA synthesis in that the product of gene 32 is essential for DNA replication, E. coli HMS174/pGP2.5-1 infected with T7A2.5: :trxA recombination, and repair (38-42). It binds cooperatively to showed a pattern different from that of wild-type T7 phage single-stranded DNA, affects denaturation and renaturation infection in that the onset ofDNA synthesis was delayed and ofDNA, and interacts specifically with T4 DNA polymerase reached a maximal rate at =40 min postinfection. and the recombination proteins uvsX and uvsY (43-46). To examine T7 DNA synthesis specifically and in more Gene 2.5 protein shares some of structural features with detail, we radioactively labeled the newly synthesized DNA other SSBs (1). These proteins possess DNA binding do- by pulse-labeling with [3H]thymidine after phage infection, mains and acidic C-terminal domains that are presumed to be cleaved the DNA with the Xmn I, and involved in protein-protein interactions (47). Studies of the examined the pattern of labeling of the DNA fragments by structural features and binding properties ofgene 2.5 protein electrophoreses through an agarose gel (Fig. 3). E. coli indicate that gene 2.5 protein contains two distinctive func- HMS174 or HMS174/pGP2.5-1 cells were infected with T7 tional domains: an N-terminal single-stranded DNA binding wild-type or T7A&2.5::trxA phages during logarithmic-phase domain and a C-terminal acidic domain (11). Tyrosine resi- dues in the DNA binding domain are required for gene 2.5 at a moi 5. At 10 and 15 min postinfection, all the growth protein binding to single-stranded DNA (11). Our recent fragments of T7A2.5::trxA DNA derived from T7A2.5- studies (unpublished data) show that the C-terminal acidic infected E. coli HMS174/pGP2.5-1 cells (lanes 8 and 9) are domain of gene 2.5 protein is essential for gene 2.5 protein labeled as are those obtained from E. coli HMS174 infected function in vivo. A truncated form of the gene 2.5 protein with wild-type T7 phage (lanes 2 and 3). However, no lacking the C-terminal 21-amino acid residues cannot support radioactively labeled T7 DNA fragments were detected in E. the growth of T7 phage lacking gene 2.5. In addition, the coli HMS174 cells infected with T7A2.5 phage (lanes 5 and 6). purified truncated gene 2.5 protein can no longer form dimers In addition, the shut-off of host DNA synthesis in T7A2.5- nor can it physically interact with T7 DNA polymerase; infected cells visualized in this experiment accounts for the however, its ability to bind to single-stranded DNA is not decrease in DNA synthesis after infection. From these ex- affected. periments, we conclude that gene 2.5 is essential for T7 DNA Why is gene 2.5 protein essential for T7 growth and replication. replication? The essential nature ofthe protein does not arise Although no DNA synthesis is observed in T7A2.5-infected solely from its ability to bind to single-stranded DNA since E. cells, RNA synthesis is grossly normal (Fig. 4). The time coli SSB, a protein that has an even higher affinity for course ofthe rate ofRNA synthesis in T7A2.5 phage-infected single-stranded DNA, cannot substitute for gene 2.5 protein cells did not differ significantly from that observed in cells in vivo. Rather, we postulate that gene 2.5 protein is essential for two reasons: (i) its specific interaction with other phage- encoded replication proteins, and (ii) its role in recombina- tion. Biochemical studies on the purified gene 2.5 protein 60 have demonstrated its physical interaction with both T7 DNA c T7 Wr polymerase and T7 helicase/primase (11) and a stimulation of FIG. 4. Time course of RNA X~ 40 the activities ofthese proteins (4, 5, 10, 11, 14, 16). Precisely synthesis. The rate of RNA synthe- why these specific interactions occur in 17, however, is not sis was measured by pulse labeling 20X T7A2.5::tixA known since other SSBs can often provide similar degrees of 620 ° with [3H]uridine for 90 sec at 4-min intervals after infection of E. coli stimulation. One exception is the rather specific stimulation HMS174 cells at 30°C with T7 wild- of primer synthesis by the gene 2.5 protein (10, 16). Presum- 0 10 20 30 40 50 type (o) or T7A2.5 phage (e) at a moi ably, the specific interaction of these proteins reflects a TimeAfter Infection (min.) = 7. requirement for a highly ordered structure at the replication Downloaded by guest on September 29, 2021 Biochemistry: Kim and Richardson Proc. Natl. Acad. Sci. USA 90 (1993) 10177 fork to coordinate leading- and lagging-strand DNA synthe- 13. Huber, H. E., Bernstein, J., Nakai, H., Tabor, S. & Richardson, sis. C. C. (1988) Cancer Cells 6, 11-17. 14. Tabor, S., Huber, H. E. & Richardson, C. C. (1987)J. Biol. Chem. Gene 2.5 is required not only for DNA synthesis but also 262, 16212-16223. for T7 recombination (8, 9). In this process, it undoubtedly 15. Huber, H. E., Tabor, S. & Richardson, C. C. (1987) J. Biol. Chem. must also interact with other recombination and replication 262, 16224-16232. proteins, such as T7 DNA polymerase and primase/helicase. 16. Mendelman, L. V. & Richardson, C. C. (1991) J. Biol. Chem. 266, However, biochemical studies support a more direct role of 23240-23250. 17 gene 2.5 protein in recombination. Upon infection of E. 17. Sadowski, P. D., Bradley, W., Lee, D. & Roberts, L. (1980) in coli with 17 phage, a single-stranded DNA renaturation Molecular Mechanisms of Recombination and Genetic Recombi- nation, eds. Alberts, B. & Cox, C. F. (Academic, New York), pp. activity is induced, suggesting that 17 gene 2.5 protein is 941-952. involved in this activity (17). It has, in fact, recently been 18. Campbell, J. L., Richardson, C. C. & Studier, F. W. (1978) Proc. shown that gene 2.5 protein facilitates the renaturation of Natl. Acad. Sci. USA 75, 2276-2280. single-stranded DNA much more efficiently that does E. coli 19. Boyer, H. W. & Roulland-Dussoix, D. (1969) J. Mol. Biol. 104, SSB, recA, or T4 gene 32 protein (S. Tabor and C.C.R., 459-465. unpublished results). Such an activity is likely to be impor- 20. Himawan, J. & Richardson, C. C. (1992) Proc. Natl. Acad. Sci. tant for catalysis of homologous pairing and may provide an USA 89, 9774-9778. 21. Young, R. A. & Davies, R. W. (1983) Proc. Natl. Acad. Sci. USA explanation for the essential role of the protein in vivo in 80, 1194-1198. recombination (8, 9). 22. Miller, J. H. (1992) Experiments in Molecular Genetics (Cold Spring By what mechanism would a defect in recombination lead Harbor Lab. Press, Plainview, NY), 2nd Ed. to the severe defect in replication observed in cells infected 23. Studier, F. W. (1969) Virology 39, 562-574. with the gene 2.S 17 mutant? One possibility is that, in the 24. Studier, F. W. (1975) J. Mol. Biol. 94, 283-295. absence of gene 2.5 protein and the catalysis of homologous 25. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular pairing, recombination intermediates accumulate and are Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY). subsequently degraded. On the other hand, it is quite likely 26. Hanahan, D. (1985) in DNA Cloning, ed. Glover, D. M. (IRL, that recombination plays an essential role in T7 DNA repli- Oxford), pp. 109-135. cation. The genome of bacteriophage T7 is replicated through 27. Innis, M. A. & Gelfand, D. H. (1990) in PCR Protocols:A Guide to exclusively linear intermediates (concatemers) (48, 49). For- Methods and Applications, eds. Innis, M. A., Gelfand, D. H., mation of concatemers provides an intermediate by which the Sninsky, J. J. & White, T. J. (Academic, San Diego), pp. 3-12. 3' ends of the 17 chromosome can be replicated. Concate- 28. Dunn, J. J. & Studier, F. W. (1983) J. Mol. Biol. 166, 477-535. mers can then be processed to yield fully replicated duplex 29. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Hey- neker, H. L., Boyer, H. W., Crosa, J. H. & Falkow, S. (1977) Gene chromosomes (50, 51). All evidence indicates that concate- 2, 95-113. mers are formed in vivo by annealing single-stranded termi- 30. Tabor, S. (1990) in Current Protocols in , eds. nally redundant ends of replication intermediates (28). We Ausubel, F. A., Brent, R., Kingston, R. E., Moore, D. D., Sei- propose that the gene 2.5 protein is required for rapid and dman, J. G., Smith, J. A. & Struhl, K. (Wiley, New York), pp. efficient homologous pairing of the exposed terminal redun- 16.2.1-16.2.11. dancy in phage-infected cells. In the absence of concatemer 31. Richardson, C. C. (1966) J. Mol. Biol. 15, 49-61. may well be defective, 32. Saito, H. & Richardson, C. C. (1981) J. Virol. 37, 343-351. formation, subsequent DNA synthesis 33. Rabkin, S. & Richardson, C. C. (1990) J. Mol. Biol. 204, 903-916. terminating after replication of the parental DNA. This one 34. Meyer, R. R. & Laime, P. S. (1990) Microbiol. Rev. 54, 342-380. round of DNA synthesis would represent only 1% or less of 35. Meyer, R. R., Glassberg, J. & Kornberg, A. (1979) Proc. Natl. the total DNA synthesis usually observed in phage-infected Acad. Sci. USA 76,1702-1705. cells. In this regard, concatemer formation in phage T7- 36. Glassberg, J., Meyer, R. R. & Kornberg, A. (1979)J. Bacteriol. 140, infected cells requires recombination (52, 53). 14-19. 37. Argos, P., Tucker, T. A. & Philipson, L. (1986) Virology 149, 208-216. We thank Stanley Tabor for helpful discussions throughout this 38. Alberts, B. M. & Frey, L. (1970) Nature (London) 227, 1313-1318. project and Qingyun Liu for assisting with the design ofT7A2.5::trxA 39. Kozinski, A. & Felgenhauer, Z. Z. (1967) J. Virol. 1, 1193-1202. and for his helpful advice. This work was supported by grants from 40. Epstein, R. H., Bolle, A., Steinberg, C., Kellenberger, C., Boy de the Department of Energy (DEG02-88ER60688), the U.S. Public la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, Health Service (AI-06045), and the American Cancer Society (NP- G. H. & Lielaulis, A. (1963) Cold Spring Harbor Symp. Quant. Biol. 1U). 28, 375-394. 41. Tomizawa, J., Araku, N. & Iwama, Y. (1966) J. Mol. Biol. 21, 1. Chase, J. W. & Williams, K. R. (1986) Annu. Rev. Biochem. 55, 247-255. 103-136. 42. Wu, J.-R. & Yeh, Y.-C. (1973) J. Virol. 12, 758-765. 2. Lohman, T. M. & Bujalowski, W. (1990) in The Biology of Non- 43. Formosa, T., Burke, R. L. & Alberts, B. M. (1983) Proc. Natl. spec rc DNA-Protein Interactions, ed. Revzen, A. (CRC, Boca Acad. Sci. USA 80, 2442-2446. Raton, FL), pp. 131-171. 44. Huberman, J. A., Kornberg, A. & Alberts, B. M. (1971) J. Mol. 3. Kowalczykowski, S. C. (1991) Annu. Rev. Biophys. Chem. 20, Biol. 62, 39-52. 539-575. 45. 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Chem. 263, 9831- 51. Serwer, P., Greenhaw, G. A. & Allen, J. L. (1982) Virology 76, 9839. 263-285. 11. Kim, Y. T., Tabor, S., Churchich, J. E. & Richardson, C. C. (1992) 52. Gilbert, W. & Dressler, D. (1968) Cold Spring Harbor Symp. Quant. J. Biol. Chem. 267, 15032-15040. Biol. 33, 173-184. 12. Richardson, C. C. (1983) Cell 33, 315-317. 53. Mosig, G. (1970) Advan. Genet. 15, 1-53. Downloaded by guest on September 29, 2021