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• W2123874820 abstract "Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities. Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions. To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA. Wild-type gp2.5 and E. coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP. Neither protein inhibits the binding of gp4 to single-stranded DNA. The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-Δ26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA. Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E. coli SSB protein is highly inhibitory at relative low concentrations. Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities. Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions. To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA. Wild-type gp2.5 and E. coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP. Neither protein inhibits the binding of gp4 to single-stranded DNA. The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-Δ26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA. Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E. coli SSB protein is highly inhibitory at relative low concentrations. The single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; gp4 and gp2.5, gene 4 protein and gene 2.5 protein, respectively; WT, wild type; SSB protein, single-stranded DNA-binding protein; nt, nucleotide. -binding protein of bacteriophage T7, encoded by gene 2.5 of the phage, is essential for T7 DNA replication and hence for growth of the phage (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 (gp2.5) is one of four proteins that constitute a T7 replisome capable of mediating DNA replication in which leading and lagging strand syntheses are coordinated and the size distribution of Okazaki fragments is maintained via a replication loop formed from the lagging strand (2Lee J. Chastain 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, 3Lee J. Chastain P.D. Griffith J.D. Richardson C.C. J. Mol. Biol. 2002; 316: 19-34Crossref PubMed Scopus (56) Google Scholar). In addition to gp2.5 the replisome contains T7 gene 5 DNA polymerase, its processivity factor Escherichia coli thioredoxin, and the multifunctional T7 gene 4 helicase-primase or gp4 (4Richardson C.C. Cell. 1983; 33: 315-317Abstract Full Text PDF PubMed Scopus (98) Google Scholar). Gp2.5 is unique among these proteins in that it not only binds to ssDNA but also physically interacts with both T7 DNA polymerase and gp4 (5Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 6Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar). Whereas the consequences of the interaction of gp2.5 with ssDNA and with T7 DNA polymerase have been characterized (5Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), little is known regarding the effect of gp2.5 on the helicase and primase activities of T7 gp4. Gp2.5 belongs to a class of ubiquitous proteins that are not only essential for DNA replication but also play key roles in DNA recombination and repair (8Kim 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, 9Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (448) Google Scholar). Gp2.5, for example, is essential for recombination in T7 phage-infected cells and in vitro it mediates homologous base pairing (10Tabor S. Richardson C.C. ****. July 9, 1996; (U. S. Patent 5,534,407)Google Scholar, 11Rezende L.F. Willcox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29098-29105Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Despite a lack of sequence homology, T7 gp2.5 is both structurally and functionally similar to the extensively studied ssDNA-binding protein of E. coli (SSB protein) and the gene 32 protein of bacteriophage T4. A recent crystal structure of a truncated gp2.5 (12Hollis 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) revealed that it contains an oligosaccharide/oligonucleotide binding fold conserved among members of the SSB protein family including E. coli SSB protein and T4 gene 32 protein. Although structurally and functionally similar, neither E. coli SSB protein nor T4 gene 32 protein can substitute T7 gp2.5 for T7 growth (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar, 13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). However, the functional interactions of gp2.5 with ssDNA and with T7 DNA polymerase and gp4 that account for this specificity have been difficult to dissect. First to be addressed is the ability of each of the latter two proteins to load onto ssDNA coated with gp2.5. T7 gp2.5 binds to ssDNA with a 10-fold lower affinity (Kd = 4.6 × 10-6 m) than does E. coli SSB protein or T4 gene 32 protein (8Kim 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). Nonetheless, T7 DNA polymerase readily extends a primer on ssDNA coated with gp2.5, E. coli SSB protein, or T4 gene 32 protein (5Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar). On the other hand, gp4 cannot mediate strand transfer in the presence of gene 32 protein, presumably because it can not load onto ssDNA coated with gene 32 protein (14Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Strand transfer mediated by the gene 4 helicase readily occurs in the presence of gp2.5 (14Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). T7 gp2.5, E. coli SSB protein, and T4 gene 32 protein all have an acidic C terminus of which a number of studies shown are essential for the protein-protein interactions observed in vitro (6Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar, 7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 15Curth U. Genschel J. Urbanke C. Greipel J. Nucleic Acids Res. 1996; 24: 2706-2711Crossref PubMed Scopus (118) Google Scholar, 16Genschel J. Curth U. Urbanke C. Biol. Chem. Hoppe-Seyler. 2000; 381: 183-192Crossref PubMed Scopus (101) Google Scholar, 17Burke R.L. Alberts B.M. Hosoda J. J. Biol. Chem. 1980; 255: 11484-11493Abstract Full Text PDF PubMed Google Scholar, 18Reddy M.S. Guhan N. Muniyappa K. J. Biol. Chem. 2001; 276: 45959-45968Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar). A truncated gp2.5, gp2.5-Δ21C, lacking the 21 C-terminal residues cannot support T7 growth, and the purified protein cannot form dimers or physically interact with T7 DNA polymerase or gp4 (6Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar). Whereas the C termini of these proteins are essential for protein-protein interactions, they are not responsible for the specificity observed for T7 growth. Chimeric proteins in which the acidic C termini of either E. coli SSB protein or T4 gene 32 protein were substituted for the C terminus of gp2.5 support the growth of T7 phage lacking gene 2.5. Furthermore, they dimerized and physically interacted with gp4 and T7 DNA polymerase. On the other hand, chimeric proteins consisting of SSB protein or gene 32 protein bearing the T7 acidic terminus could not support the growth of T7 lacking gene 2.5 (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Using a random mutagenesis procedure, we recently isolated a number of gp2.5s having single amino acid substitutions that could not support T7 growth (20Rezende 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). Whereas studies with these altered proteins led to the identification of sites that affected DNA binding (7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 21Hyland E.M. Rezende L.R. Richardson C.C. J. Biol. Chem. 2003; 278: 7247-7256Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and homologous base pairing (11Rezende L.F. Willcox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29098-29105Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), none of the altered proteins appeared to be defective in interactions with gp4 or T7 DNA polymerase. One altered protein, gp2.5-F232L, identified in the study, had a single amino acid substitution in the C-terminal residue (20Rezende 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). Gp2.5-F232L binds more tightly to ssDNA (Kd = 1.5 × 10-6 m) than does WT gp2.5 but not as tightly as gp2.5-Δ21C (Kd = 1.1 × 10-7 m) (7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Gp2.5-F232L stimulates T7 DNA polymerase activity on ssDNA even more so than does WT gp2.5, whereas gp2.5-Δ21C inhibits T7 DNA polymerase activity. This result clearly shows the importance of the C-terminal tail in interactions with the polymerase. Less well studied are the effects of gp2.5 on the helicase and primase activities of the T7 gp4. The helicase and primase activities of gp4 reside in the C-terminal and N-terminal halves of the 63-kDa protein, respectively (22Mendelman L.V. Notarnicola S.M. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10638-10642Crossref PubMed Scopus (43) Google Scholar). The DNA sequence encoding each domain has been independently cloned, and the respective helicase and primase fragments have been overproduced and purified (22Mendelman L.V. Notarnicola S.M. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10638-10642Crossref PubMed Scopus (43) Google Scholar). Crystal structures of both the helicase (23Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 24Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar) and primase (25Kato M. Ito T. Wagner G. Richardson C.C. Ellenberger T. Mol. Cell. 2003; 111: 1349-1360Abstract Full Text Full Text PDF Scopus (90) Google Scholar) fragments are available. The helicase and primase fragments have helicase and primase activities, respectively, equivalent to that of the wild-type full-length gp4. However, as discussed below, the helicase domain, when covalently attached to the primase domain as in the full-length protein, bestows several desirable properties on the primase domain. The multiple activities catalyzed by the gp4, many of which involve interactions with ssDNA, likewise provide multiple reactions that are potential targets for gp2.5. For this reason, it is important to review briefly the reactions mediated by each domain of the gp4. Reactions mediated by the C-terminal helicase domain require the assembly of the protein into a hexamer. In the presence of dTTP, the 63-kDa gp4 assembles as a hexamer on ssDNA (26Egelman E.H. Yu X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (252) Google Scholar, 27Yu X. Hingorani M.M. Patel S.S. Egelman E.H. Nat. Struct. Biol. 1996; 3: 740-742Crossref PubMed Scopus (115) Google Scholar) with a polarity such that the helicase domain faces the 3′-end of the DNA to which it is bound (26Egelman E.H. Yu X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (252) Google Scholar). A major stabilization of the hexamer is dependent on the linker region that connects the helicase and primase domains (23Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 24Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 28Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Once bound to ssDNA, the protein then translocates unidirectionally from 5′ to 3′ on the strand, a reaction coupled to the hydrolysis of dTTP (29Kim D.E. Narayan M. Patel S.S. J. Mol. Biol. 2002; 321: 807-819Crossref PubMed Scopus (85) Google Scholar, 30Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 205-209Crossref PubMed Scopus (107) Google Scholar). Upon encountering a duplex provided that there is a 3′-ssDNA tail on the strand that it encounters, it will unwind the duplex. Gp2.5 bound to ssDNA and/or to the gp4 could facilitate the loading of the hexamer onto ssDNA. Unlike the DnaB helicase of E. coli or the gene 41 helicase of phage T4 (31Hinton D.M. Silver L.L. Nossal N.G. J. Biol. Chem. 1985; 260: 12851-12857Abstract Full Text PDF PubMed Google Scholar, 32Barry J. Albert B. J. Biol. Chem. 1994; 269: 330-362Google Scholar), the T7 helicase does not have a specific helicase loader. Once loaded onto ssDNA, the presence of gp2.5-coated ssDNA could conceivably facilitate or hinder translocation of the gp4. The N-terminal primase domain catalyzes the template-directed synthesis of oligoribonucleotides on ssDNA, and these, in turn, are transferred to the T7 DNA polymerase for use as primers to initiate DNA synthesis (33Frick D.N. Richardson C.C. Annu. Rev. Biochem. 2001; 70: 39-80Crossref PubMed Scopus (305) Google Scholar). At the basic primase recognition site, 5′-GTC-3′, the primase catalyzes the synthesis of the dinucleotides pppAC from ATP and CTP (34Mendelman L.V. Richardson C.C. J. Biol. Chem. 1991; 266: 23240-23250Abstract Full Text PDF PubMed Google Scholar). Provided that the more extensive recognition sites are present, 5′-GGGTC-3′, 5′-TGGTC-3′, or 5′-GTGTC-3′ and the functional tetraribonucleotide primers, pppACCC, pppACCA, and pp-pACAC are synthesized (30Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 205-209Crossref PubMed Scopus (107) Google Scholar). A major role in sequence recognition is mediated by a Cys4 zinc motif located at the extreme N terminus of the primase domain (5Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 34Mendelman L.V. Richardson C.C. J. Biol. Chem. 1991; 266: 23240-23250Abstract Full Text PDF PubMed Google Scholar, 35Bernstein J.A. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 85: 396-400Crossref Scopus (93) Google Scholar). Obviously, the effect of gp2.5-coated DNA on the recognition of the primase sites and the subsequent synthesis of the primer are of considerable importance. The primase fragment lacking the helicase domain catalyzes the template-directed synthesis of oligoribonucleotides equally as well as does the full-length gp4 in the absence of dTTP (36Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar). However, the primase fragment alone has a very low affinity for ssDNA and for its recognition site (37Frick D.N. Richardson C.C. J. Biol. Chem. 1999; 274: 35889-35898Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Consequently, the 63-kDa gp4 has markedly higher primase activity in the presence of dTTP because of the ability of the hexameric helicase to bind tightly to ssDNA (36Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar). On long DNA templates with a scarcity of primase recognition sites, the primase is dependent on translocation by the helicase domain for delivery to these sites (36Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar). As a result, gp2.5 could have an indirect effect on primase activity via its interaction with the helicase domain. In a recent paper (7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), we examined the effect of wild-type gp2.5 and variants of gp2.5 for their ability to bind to ssDNA and to interact with T7 DNA polymerase. Wild-type gp2.5 was found to slightly stimulate DNA synthesis catalyzed by T7 DNA polymerase on ssDNA templates. The ability of T7 DNA polymerase to catalyze synthesis through gp2.5-coated DNA is dependent on the presence of the acidic C-terminal tail in that gp2.5-Δ26C lacking the 26 C-terminal amino acids inhibits synthesis by >10-fold (7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In this communication, we examined the effect of gp2.5 on the multiple activities catalyzed by the helicase and primase domains of gp4. In these studies, we have used two altered gp2.5s, gp2.5-Δ26C and gp2.5-F232L, as well as the wild-type gp2.5. Gp2.5-ΔF232L has a single amino acid substitution in the C-terminal tail that increases its binding to ssDNA yet does not affect its interaction with the T7 replication proteins (7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In addition, we have examined the effect of E. coli SSB protein on these reactions to examine the specificity for gp2.5 in the T7 system. Bacterial Strains, Bacteriophages, and Plasmids—E. coli BL21(DE3)-(F-ompT hsdSB (rB-mB-) gal-dcm (DE3)) (Novagen) was used as the host strain to express T7 gene 2.5 and to purify wild-type and mutant gp2.5. Wild-type and mutant gene 2.5 are expressed from the pET17b plasmid (Novagen) containing the T7 RNA polymerase promoter. T7 gp2.5-Δ26C was obtained from Edel Hyland (Harvard Medical School). E. coli HMS 174 (DE3) was purchased from Invitrogen. DNA and Oligonucleotides—Oligonucleotides (Table I) used in the assay to measure primase activity (ZHP20, ZHP70, and ZH70) and to prepare helicase substrates (S75 and L95) were purchased from Integrate DNA Technologies. [5′-32P]S75 oligonucleotide was annealed to L95 and used for helicase analysis substrates. M13 ssDNA was purchased from Invitrogen. The oligonucleotides used for cloning of gene 2.5 were described previously (7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar).Table IOligonucleotides (Oligo) and their sequencesOligoSequence (5′→3′)Underlined sequenceS75aS75 and L95 oligonucleotides were annealed to generate the DNA substrate for measuring helicase activityCGCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACATGCTTTTTTTTTTTTTTTTTTTTAnnealed nucleotidesL95aS75 and L95 oligonucleotides were annealed to generate the DNA substrate for measuring helicase activityTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCATGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGCGAnnealed nucleotidesZH70TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGTCCCCCCCCCCCCCCCCPimase siteZHP20GTGTCTTTTTTTTTTTTTTTPrimase siteZHP70TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCGTGTCTTTTTTTTTTTTTTTPrimase sitea S75 and L95 oligonucleotides were annealed to generate the DNA substrate for measuring helicase activity Open table in a new tab Proteins, Enzymes, and Chemicals—Restriction enzymes, T4 polynucleotide kinase, T4 DNA ligase, and calf-intestinal phosphatase were purchased from New England Biolabs. E. coli SSB protein was purchased from United States Biochemicals Corp. Donald Crampton (Harvard Medical School) supplied T7 gp4, and Seung-Joo Lee (Harvard Medical School) supplied the primase fragment of T7 gp4. All of the chemicals and reagents were from Sigma unless otherwise noted. Protein Overexpression and Purification—WT gp2.5 and gp2.5-F232L were overexpressed and purified as described previously (7He Z.G. Rezende L.F. Willox S. Griffith J.D. Richardson C.C. J. Biol. Chem. 2003; 278: 29538-29545Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Oligoribonucleotide Synthesis by T7 DNA Primase—The synthesis of oligoribonucleotides catalyzed by gp4 was determined by measuring the incorporation of [α-32P]CMP into oligoribonucleotides using M13 ssDNA or synthetic DNA templates (Table I) containing a primase recognition site (34Mendelman L.V. Richardson C.C. J. Biol. Chem. 1991; 266: 23240-23250Abstract Full Text PDF PubMed Google Scholar, 37Frick D.N. Richardson C.C. J. Biol. Chem. 1999; 274: 35889-35898Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The reaction (10 μl) included 4 nm M13 ssDNA or 400 nm oligonucleotide, 0.3 mm each of ATP and [α-32P]CTP (0.1 μCi), 0.5 mm dTTP, 40 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 10 mm dithiothreitol, 50 mm potassium glutamate, and 100 μg/ml bovine serum albumin. The reaction mixtures were preincubated with the indicated amount of ssDNA-binding proteins for 5 min at 37 °C, and the reactions were initiated by the addition of either 80 nm gp4 monomer or 800 nm gp4 primase fragment. After further incubation at 37 °C for 30 min, the reaction was terminated by the addition of 3 μl of sequencing dye (98% formamide, 10 mm EDTA, pH 8.0, 0.1% xylene cyanol FF, and 0.1% bromphenol blue) and loaded onto a 25% denaturing polyacrylamide sequencing gel containing 3 m urea. After electrophoresis, the radioactive oligoribonucleotide products were analyzed using a Fuji BAS 1000 Bioimaging analyzer. dTTPase Assay—Gp4 catalyzes the ssDNA-dependent hydrolysis of dTTP, a reaction coupled to its translocation on ssDNA (38Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar). The reaction (10 μl) contained 1.2 nm M13 ssDNA, 0.5 mm [α-32P]dTTP (0.1 μCi), 40 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 10mm dithiothreitol, and 50 mm potassium glutamate. The reaction mixtures were preincubated with the indicated amount of ssDNA-binding protein for 5 min at 37 °C, and the reactions were initiated by the addition of 80 nm gp4. After further incubation at 37 °C for 30 min, the reactions were terminated by adding EDTA to a final concentration of 25 mm. The reaction mixture was spotted onto a polyethyleneimine cellulose thin layer chromatography (TLC) plate. The TLC plate was developed with a solution containing 1 m formic acid and 0.8 m LiCl. The TLC plate was analyzed using a Fuji BAS 1000 Bioimaging analyzer. DNA Unwinding Assay—The assay for helicase activity measures the release of a radioactively labeled oligonucleotide partially annealed to a complementary ssDNA (39Matson S.W. Tabor S. Richardson C.C. J. Biol. Chem. 1983; 258: 14017-14024Abstract Full Text PDF PubMed Google Scholar). A helicase substrate was prepared by annealing a 5′-end-labeled 75-mer oligonucleotide S75 (Table I) to a 95-mer oligonucleotide L95 (Table I) in 0.1 m NaCl. The helicase reaction (10 μl) contained 60 nm DNA substrate, 1 mm dTTP, 40 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 10 mm dithiothreitol, and 50 mm potassium glutamate. The reaction mixtures were preincubated with the indicated amount of ssDNA-binding protein for 5 min at 37 °C, and the reactions were initiated by the addition of 80 nm gp4. After further incubation for 10 min at 37 °C, the reaction was terminated by adding EDTA to a final concentration of 25 mm. The reaction mixture was loaded onto a 10% denaturing polyacrylamide gel. After electrophoresis, the radioactive oligonucleotide disassociated from the partial duplex was measured using a Fuji BAS 1000 Bioimaging analyzer. In a control experiment in which 60 nm each of ssDNA 75 and 95-mer were incubated, we found that there is significant annealing (∼30%) of the DNA strands. Therefore, it is likely that some reannealing of ssDNA strands arising during the helicase reaction occurs, thus leading to an underestimation of DNA unwinding. Effect of Gp2.5 and E. coli SSB Protein on Primer Synthesis—An earlier study using a gp4 preparation enriched for the 63-kDa species showed that gp2.5-stimulated gp4 catalyzed oligoribonucleotide synthesis on M13 ssDNA 3.5-fold (40Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar). Subsequent studies confirmed this result and also revealed that gp2.5-Δ21C, lacking its C-terminal 21 amino acids, severely inhibited oligoribonucleotide synthesis (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In that study, E. coli SSB protein stimulated oligoribonucleotide synthesis ∼2-fold, whereas T4 gp32 protein inhibited synthesis by >10-fold (13Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To further characterize these interactions, we examined the ability of WT gp2.5 and E. coli SSB protein to affect gp4-catalyzed primer synthesis at various concentrations. Primase activity was assayed using M13 ssDNA coated with either WT gp2.5 or E. coli SSB protein. Consistent with the previous studies, both gp2.5 and E. coli SSB protein stimulated oligoribonucleotide synthesis ∼2-3 fold (Fig. 1A). Our earlier studies using gel shift analysis have shown that 1 μm gp2.5 is sufficient to coat 3.3 nm 70-mer ssDNA (21Hyland E.M. Rezende L.R. Richardson C.C. J. Biol. Chem. 2003; 278: 7247-7256Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In this experiment containing 4 nm M13 ssDNA, we used a gp2.5 concentration of 20 μm, which is sufficient to coat 20 nm M13 ssDNA based on the assumption that one gp2.5 molecule coats 6-7 nucleotides on ssDNA (8Kim 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). E. coli SSB protein has approximately a 10-fold higher affinity for ssDNA relative to gp2.5 (8Kim 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). Interestingly, maximal stimulation occurred with the lowest amount of gp2.5 (2 μm), an amount sufficient to coat 50% of the ssDNA. E. coli SSB protein stimulated oligoribonucleotide synthesis at an even lower concentration of 0.2 μm (Fig. 1A). To eliminate the effect of secondary structure in the M13 DNA substrate, we used a 70-mer oligonucleotide, ZH70, containing only one primase site as a template (Table I) to analyze primer synthesis. For the assay, an increasing concentration of ssDNA-binding protein was present to coat the ssDNA in the reaction. Consistent with the results obtained with M13 ssDNA, both WT gp2.5 and E. coli SSB protein stimulated the primase activity of gp4 at low concentrations (Fig. 1B). Effect of Gp2.5 and E. coli SSB Protein on Binding of Gp4 to ssDNA—The first step of primer synthesis is the loading of gp4 onto the ssDNA template. T" @default.
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• W2123874820 date "2004-05-01" @default.
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• W2123874820 title "Effect of Single-stranded DNA-binding Proteins on the Helicase and Primase Activities of the Bacteriophage T7 Gene 4 Protein" @default.
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• W2123874820 doi "https://doi.org/10.1074/jbc.m401100200" @default.
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