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W2036768907 abstract "The functional role of the φ29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage φ29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the φ29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the φ29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching φ29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in φ29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages. The functional role of the φ29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage φ29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the φ29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the φ29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching φ29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in φ29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages. Studies on DNA replication and related processes have provided detailed insights in the function of many proteins involved in these processes (for review, see Ref. 1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman and Co., San Fransisco1992Google Scholar). Despite this, little is known about the in vivo organization of DNA replication. To gain a better insight in this fundamental process, we studied the in vivo DNA replication of the Bacillus subtilisbacteriophage φ29 (2Meijer W.J.J. Lewis P.J. Errington J. Salas M. EMBO J. 2000; 19: 4182-4190Crossref PubMed Scopus (18) Google Scholar). The detailed knowledge of its in vitro mechanism of DNA replication (for reviews, see Refs. 3Salas M. Rojo F. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. American Society for Microbiology, Washington, D. C.1993: 843-858Google Scholar and4Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 131-176Google Scholar) made φ29 an attractive system for this study. The genome of φ29 is a linear double-stranded DNA (dsDNA) 1dsDNAdouble-stranded DNAssDNAsingle-stranded DNATPterminal proteinSSBsingle-stranded DNA-binding protein 1dsDNAdouble-stranded DNAssDNAsingle-stranded DNATPterminal proteinSSBsingle-stranded DNA-binding protein of 19,285 base pairs that contains a terminal protein (TP) covalently linked at each 5′ end. Fig. 1 A shows a schematic representation of the genetic and transcriptional organization of the φ29 genome. Regulation of φ29 DNA transcription, which can be divided into an early and a late stage, has been studied extensivelyin vivo as well as in vitro (for reviews, see Refs. 3Salas M. Rojo F. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. American Society for Microbiology, Washington, D. C.1993: 843-858Google Scholar and 5Rojo F. Mencı́a M. Monsalve M. Salas M. Prog. Nucleic Acid Res. Mol. Biol. 1998; 60: 29-46Crossref PubMed Scopus (33) Google Scholar). The late expressed genes, all transcribed from a single operon present in the central part of the genome, encode the structural proteins of the phage, proteins involved in morphogenesis, and those required for lysis of the infected cell. The early expressed genes are present in two operons that flank the late operon. The early operon located at the left side of the φ29 genome encodes the transcriptional regulator protein p4 and various proteins that are directly involved in phage DNA replication, such as the DNA polymerase, TP, single-stranded DNA-binding protein (SSB), double-stranded DNA-binding protein, and protein p1. The operon located at the right side of the φ29 genome encodes, in addition to proteins p17 and p16.7, four putative proteins of unknown function. double-stranded DNA single-stranded DNA terminal protein single-stranded DNA-binding protein double-stranded DNA single-stranded DNA terminal protein single-stranded DNA-binding protein A schematic overview of the in vitro φ29 DNA replication mechanism is shown in Fig. 1 B. Initiation of φ29 DNA replication occurs via a so-called protein-primed mechanism (reviewed in Refs. 3Salas M. Rojo F. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. American Society for Microbiology, Washington, D. C.1993: 843-858Google Scholar, 4Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 131-176Google Scholar, and 6Meijer W.J.J. Horcajadas J.A. Salas M. Microbiol. Mol. Biol. Rev. 2001; 65: 261-287Crossref PubMed Scopus (156) Google Scholar). The TP-containing DNA ends constitute the origins of replication. Initiation of DNA replication starts by recognition of the origin by a heterodimer formed by the φ29 DNA polymerase and the primer TP. The DNA polymerase then catalyzes the addition of the first dAMP to the primer TP. Next, after a transition step, these two proteins dissociate, and the DNA polymerase continues processive elongation until replication of the nascent DNA strand is completed. Replication, which starts at both DNA ends, is coupled to strand displacement. This results in the generation of so-called type I replication intermediates consisting of full-length double-stranded φ29 DNA molecules with one or more single-stranded DNA (ssDNA) branches of varying lengths. When the two converging DNA polymerases merge, a type I replication intermediate becomes physically separated into two type II replication intermediates. Each of these consists of a full-length φ29 DNA molecule in which a portion of the DNA, starting from one end, is double-stranded, and the portion spanning to the other end is single-stranded. Over the last decades convincing evidence has been presented that replication of bacterial genomes, including that of resident plasmids and infecting phages, occurs at the bacterial cell membrane (for review, see Ref. 7Firshein W. Annu. Rev. Microbiol. 1989; 43: 89-120Crossref PubMed Scopus (75) Google Scholar). Also, replication of φ29 DNA takes place at the membrane of the infected cell (2Meijer W.J.J. Lewis P.J. Errington J. Salas M. EMBO J. 2000; 19: 4182-4190Crossref PubMed Scopus (18) Google Scholar, 8Ivarie R.D. Pène J.J. Virology. 1973; 52: 351-362Crossref PubMed Scopus (26) Google Scholar, 9Bravo A. Salas M. J. Mol. Biol. 1997; 269: 102-112Crossref PubMed Scopus (36) Google Scholar). Gene 16.7, present in the early expressed operon located at the right side of the φ29 genome (see Fig. 1 A), encodes an integral membrane protein of 130 amino acids. The efficiency of in vivo φ29 DNA replication is affected in the absence of protein p16.7 (10Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar). In this work we analyzed the functional role of p16.7 in φ29 DNA replication using purified p16.7A, a soluble variant of p16.7 lacking the N-terminal transmembrane-spanning domain. We found that protein p16.7A can functionally substitute the φ29 SSB p5 in in vitroφ29 DNA amplification assays, suggesting that it is a ssDNA-binding protein. This inference was further supported by direct assays such as electron microscopy and gel retardation studies. Thus, in addition to a classical SSB p5, φ29 encodes a membrane-localized ssDNA-binding protein. Contrary to the SSB p5, p16.7A has no helix-destabilizing activity, and p16.7 is not synthesized in stoichiometric amounts in infected cells. These and other results show that p16.7 and SSB p5 have non-overlapping functions. Based on the properties determined in this work together with the features described before, it is most likely that p16.7 attaches φ29 DNA to the membrane of the infected cells by binding to the stretches of ssDNA present in the replication intermediates. B. subtilis 110NA (trpC2, spoOA3,su− (11Moreno F. Camacho A. Viñuela E. Salas M. Virology. 1974; 62: 1-16Crossref PubMed Scopus (69) Google Scholar)) was used as the non-suppressor strain for φ29 infections. Cells, grown at 37 °C in LB medium supplemented with 5 mm MgSO4, were infected with phage φ29 mutant sus14(1242) (12Jiménez F. Camacho A. de la Torre J. Viñuela E. Salas M. Eur. J. Biochem. 1977; 73: 57-72Crossref PubMed Scopus (43) Google Scholar) at a multiplicity of infection of 5. Phage φ29 sus14(1242) contains a suppressor-sensitive mutation in gene 14 that encodes the holin gene. As a consequence, cell lysis is delayed, which allowed determination of the amounts of p16.7 and SSB at late stages in the infection cycle. The mutation has no effect on phage DNA replication or phage morphogenesis and, therefore, is considered as wild-type phage in these studies. Escherichia coli strain JM109 (F′ traD36 lacIqΔ(lacZ)M15 proA+B+/e14 −(McrA −) Δ(lac-proAB) thi gyrA96(Nalr) endA1 hsdR17(rk − mk+) relA1 supE44 recA1) harboring plasmid pUSH16.7A (10Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar) was used for overexpression of protein p16.7A. All DNA manipulations were carried out according to Sambrook et al. (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). [α-32P]ATP and [γ-32P]ATP (3000 Ci/mmol) were obtained from Amersham Biosciences, Inc. DNA fragments were isolated from agarose gels using the Qiaex gel extraction kit (Qiagen, Inc., Chatsworth, CA). PCR reactions were carried out with proofreading-proficient Vent DNA polymerase (New England Biolabs, Beverly, MA) using conditions as described before (10Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar). Oligonucleotides were purchased from Isogen Bioscience BV (Maarsen, The Netherlands). Protein p16.7A was overexpressed and purified using a Ni2+-nitrilotriacetic acid resin column as described before (10Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar). These assays were performed as described before (14Serna-Rico A. Illana B. Salas M. Meijer W.J.J. J. Biol. Chem. 2000; 275: 40529-40538Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The reaction mixtures of the φ29 TP-DNA replication assays contained the indicated amount of protein p16.7A or SSB p5. The amplification assays were stopped after an incubation period of 20 min. Replication reactions were carried out in the absence or in the presence of p16.7A or SSB p5 as described below. After 30 min at 30 °C the samples were stopped by adding 0.05 volumes of 4,5′,8-trimethylpsoralen (200 μg/ml in 100% ethanol) on ice. The samples were then irradiated with 366-nm UV light on ice for 1 h with 2 psoralen additions, as described by Sogo and Thoma (15Sogo J.M. Thoma F. Methods Enzymol. 1989; 170: 142-165Crossref PubMed Scopus (41) Google Scholar). These cross-linking conditions were sufficient to produce essentially complete cross-linking of the DNA molecules in the absence of protein. After psoralen cross-linking, the samples were digested with proteinase K (500 μg/ml) for 2 h at 56 °C and extracted with phenol, and the DNA was precipitated with ethanol. Denaturation and spreading of the psoralen cross-linked DNA for electron microscopy were carried out according the BAC technique, as described by Sogoet al. (16Sogo J.M. Stasiak A. DeBernadin W. Losa R. Koller T. Sommerville J. Scheer U. Electron Microscopy in Molecular Biology. A Practical Approach. IRL Press at Oxford University Press, Oxford1987: 61-79Google Scholar). Electron micrographs were taken with a Philips 420 electron microscope at 80 kV, routinely at a magnification of 20,000-fold. Unless stated otherwise, the incubation mixtures contained, in a final volume of 20 μl, 25 mm Hepes, pH 7.5, 4% Ficoll 400, 1 mm EDTA, 0.1 mg/ml bovine serum albumin, 10 mm dithiothreitol, the indicated labeled DNA fragment, and the indicated amount of protein p16.7A or φ29 SSB p5. After incubation for 10 min at 4 °C, the samples were subjected to electrophoresis in 4% non-denaturing polyacrylamide (80:1) gels containing 12 mm Tris acetate, pH 7.5, and 1 mm EDTA and run at 4 °C using a running buffer containing 12 mm Tris acetate, pH 7.5, and 1 mm EDTA at 70 V for 6 h. Next, the gels were dried and autoradiographed. Twenty μg of protein p16.7A was subjected to a 15–30% linear glycerol gradient containing 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, and 7 mm β-mercaptoethanol and run as described before (14Serna-Rico A. Illana B. Salas M. Meijer W.J.J. J. Biol. Chem. 2000; 275: 40529-40538Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). After fractionation of the gradient, aliquots of each fraction were analyzed by SDS-PAGE and gel retardation assays. Oligonucleotide degradation assays and the helix destabilization assays, the latter using 125 ng of primed M13mp18 DNA, were carried out as described by Gascón et al. (17Gascón I. Lázaro J.M. Salas M. Nucleic Acids Res. 2000; 28: 2034-2042Crossref PubMed Google Scholar). Oligonucleotide −40 Universal and the same with a 5′ (5 thymidines) or a 3′ (5 adenines) extension were end-labeled with T4-polynucleotide kinase using [γ-32P]ATP for 1 h at 37 °C. To determine the intracellular levels of the viral proteins p16.7 and SSB throughout the course of the φ29 infection cycle, B. subtilis cells were infected with phage φ29 mutant sus14(1242) as described above. At different times after infection, 1.5-ml samples were withdrawn and processed as described before (10Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar). Known amounts (ng) of the corresponding purified proteins (p16.7A or SSB p5) were run in the same gel to determine the standard curve. Polyclonal antisera from rabbits against φ29 p16.7 or against φ29 SSB p5 were diluted 2,500 and 1,000 times, respectively. Previous results show that the absence of protein p16.7 affects the efficiency of in vivo φ29 DNA replication, especially at early infection times (10Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar). Protein p16.7 might stimulate φ29 DNA replication by enhancing the rate-limiting step of initiation of DNA replication. To study this possibilityin vitro, φ29 DNA replication initiation assays were performed in the absence or presence of increasing amounts of purified p16.7A. Although efficient initiation requires the presence of φ29 template TP-DNA, the DNA polymerase is also able to carry out the TP-deoxynucleotidylation reaction in the absence of TP-DNA (18Blanco L. Bernad A. Esteban J.A. Salas M. J. Biol. Chem. 1992; 267: 1225-1230Abstract Full Text PDF PubMed Google Scholar). Therefore, in vitro φ29 DNA initiation experiments were performed in reaction mixtures either lacking or containing template TP-DNA and in the absence or presence of increasing amounts of protein p16.7A. No significant effect of p16.7A on the initiation reactions was obtained (not shown), indicating that p16.7A plays no role in thein vitro initiation of φ29 DNA replication. The possibility that p16.7A has a role in φ29 DNA replication after the initiation step was analyzed by studying the possible effects of protein p16.7A in an in vitro φ29 DNA amplification system. This system, which allows the amplification of very low amounts of φ29 template DNA, requires the following four φ29-encoded proteins: DNA polymerase, TP, double-stranded DNA-binding protein p6, and the SSB p5 (19Blanco L. Lázaro J.M. de Vega M. Bonnin A. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12198-12202Crossref PubMed Scopus (74) Google Scholar). It has been described that omission of SSB p5 from the reaction mixtures results in the generation (19Blanco L. Lázaro J.M. de Vega M. Bonnin A. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12198-12202Crossref PubMed Scopus (74) Google Scholar) and amplification (20Esteban J.A. Blanco L. Villar L. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2921-2926Crossref PubMed Scopus (12) Google Scholar) of short φ29 DNA products. Esteban et al. (20Esteban J.A. Blanco L. Villar L. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2921-2926Crossref PubMed Scopus (12) Google Scholar) demonstrated that the short φ29 DNA products are of palindromic nature and that they are caused by a DNA polymerase template-switching event during replication. Binding of SSB p5 to the displaced stretches of ssDNA present in type I DNA replication intermediates avoids the DNA polymerase to switch template, thus preventing the generation of short DNA products. Interestingly, we found that the addition of increasing amounts of protein p16.7A to reaction mixtures lacking SSB p5 resulted in the synthesis of increasing amounts of full-sized φ29 DNA and a concomitant decrease in the amounts of short DNA products (Fig.2). In fact, the generation of the short DNA products was fully prevented when the reaction mixtures contained 14 or 28 μm protein p16.7A. These results show that, under the conditions tested, the presence of protein p16.7A prevents the DNA polymerase from switching template and, therefore, that it can functionally substitute the SSB p5. The most likely explanation for these results is that the p16.7A protein binds to the stretches of displaced ssDNA present in the type I replication intermediates. The amount of full-sized φ29 DNA synthesized in the presence of 14 or 28 μm protein p16.7A is slightly lower than that compared in the presence of 7 μm. Probably, this decrease is due to binding of protein p16.7A to double-stranded φ29 template DNA at these elevated p16.7A concentrations (see below), causing a small effect on the efficiency of the φ29 DNA amplification. Psoralen can cross-link portions of a dsDNA molecule that are free of protein (15Sogo J.M. Thoma F. Methods Enzymol. 1989; 170: 142-165Crossref PubMed Scopus (41) Google Scholar) as well as folded-back regions in ssDNA (21Wollenzien P.L. Gasparro F.P. Psoralen-DNA Photobiology. CRC Press, Inc., Boca Raton, FL1988: 51-85Google Scholar). The presence of the φ29 SSB p5 was shown to prevent the displaced ssDNA of the φ29 DNA replication intermediates from psoralen cross-linking, demonstrating that it binds to ssDNA (22Gutiérrez C. Sogo J.M. Salas M. J. Mol. Biol. 1991; 222: 983-994Crossref PubMed Scopus (22) Google Scholar, 23Gutiérrez C. Martı́n G. Sogo J.M. Salas M. J. Biol. Chem. 1991; 266: 2104-2111Abstract Full Text PDF PubMed Google Scholar, 24Soengas M.S. Gutiérrez C. Salas M. J. Mol. Biol. 1995; 253: 517-529Crossref PubMed Scopus (66) Google Scholar). To study whether protein p16.7A indeed binds to the ssDNA portions of φ29 DNA replication intermediates, as indicated by the results of the φ29 DNA amplification assays described above, we used the psoralen cross-linking technique. Thus, φ29 DNA replication reactions, carried out in the absence or presence of either protein p16.7A or SSB p5, were treated with psoralen as described under “Materials and Methods.” Next, after the reaction products were treated with proteinase K, extracted with phenol, and purified, they were spread under denaturing conditions and analyzed by electron microscopy. As expected, in all three samples full-length dsDNA molecules with one or more ssDNA tails (type I) and full-length DNA molecules formed by a dsDNA portion of variable length from one DNA end plus an ssDNA portion spanning to the other DNA end (type II) were observed. A representative type I replication intermediate of each sample is shown in Fig.3. As described before (22Gutiérrez C. Sogo J.M. Salas M. J. Mol. Biol. 1991; 222: 983-994Crossref PubMed Scopus (22) Google Scholar, 23Gutiérrez C. Martı́n G. Sogo J.M. Salas M. J. Biol. Chem. 1991; 266: 2104-2111Abstract Full Text PDF PubMed Google Scholar, 24Soengas M.S. Gutiérrez C. Salas M. J. Mol. Biol. 1995; 253: 517-529Crossref PubMed Scopus (66) Google Scholar), the displaced ssDNA regions of replication intermediates produced in the absence (Fig. 3 A) or the presence of SSB p5 (Fig.3 C) appeared as collapsed and well unfolded structures, respectively. Fig. 3 B shows that the ssDNA portions in replication intermediates produced in the presence of protein p16.7A also had a well unfolded structure. These results, therefore, show that protein p16.7A, like the SSB p5, prevents the ssDNA from psoralen cross-linking, demonstrating that it binds to the ssDNA portions of the replication intermediates generated during φ29 DNA replication. Binding of p16.7A to ssDNA was further analyzed by gel mobility shift assays. For this purpose, the 175-base pair right-end fragment of the φ29 genome was end-labeled with 32P (see “Materials and Methods”), heat-denatured, and used in gel retardation assays in the absence or presence of increasing amounts of purified protein p16.7A. Although retardation of the ssDNA fragment was observed in the presence of 45 nm p16.7A, full retardation of all the ssDNA molecules required a p16.7A concentration of 360 nm (Fig. 4 A). Similar results were obtained with various other DNA fragments (results not shown). To gain an insight in the global ssDNA binding of p16.7A compared with that of the well studied φ29 protein p5, the experiment shown in Fig. 4 A was carried out in parallel using purified SSB p5. Whereas in agreement with previously published results (25Martı́n G. Lázaro J.M. Méndez E. Salas M. Nucleic Acids Res. 1989; 17: 3663-3672Crossref PubMed Scopus (40) Google Scholar, 26Soengas M.S. Esteban J.A. Salas M. Gutiérrez C. J. Mol. Biol. 1994; 239: 213-226Crossref PubMed Scopus (25) Google Scholar, 27Gascón I. Gutiérrez C. Salas M. J. Mol. Biol. 2000; 296: 989-999Crossref PubMed Scopus (16) Google Scholar), some retardation of part of the ssDNA molecules occurred in the presence of 3.3 μm SSB p5, full retardation of all the ssDNA molecules required a concentration of 13.3 μm(Fig. 4 B). Together, these results show that the ssDNA binding activity of p16.7A is about 50 times higher than that of the φ29 SSB. To study possible binding of protein p16.7A to dsDNA, the same fragment of the φ29 genome used in Fig. 4, A andB, but in its double-stranded form, was used in gel retardation assays. The results, presented in Fig. 4 C, show that p16.7A binds to dsDNA, although the amount of p16.7A required to obtain full retardation of all the dsDNA molecules was about 20-fold higher than that required to bind the same DNA molecules in their single-stranded form. The observation that a smear of retarded DNA species is observed at low p16.7A concentrations (45–180 and 180–720 nm for ss- and dsDNA, respectively) indicates that the nucleoprotein complex formed at these concentrations is rather unstable. In addition, the increasing mobility shift caused by the various concentrations of p16.7A protein analyzed suggests the binding of more than one protein molecule per DNA molecule. This inference is further supported by the findings that a considerable amount of protein p16.7A is required (i) to prevent the generation of short ssDNA products in amplification assays lacking the SSB p5 (see above) and (ii) to cover the complete circular M13 ssDNA in order to prevent binding of the φ29 DNA polymerase (see below). To confirm that the observed retardation was due to protein p16.7A and not to a possible minor contaminant, the purified p16.7A protein was subjected to a glycerol gradient, after which aliquots of the gradient were analyzed for ssDNA binding by gel retardation. The results presented in Fig. 4 D show that the ssDNA binding activity was restricted to those fractions that contained protein p16.7A. The following approach was used to study possible binding of p16.7A to circular ssDNA. The φ29 DNA polymerase has strong affinity for naked ssDNA (28Blanco L. Bernad A. Lázaro J.M. Martı́n G. Garmendia C. Salas M. J. Biol. Chem. 1989; 264: 8935-8940Abstract Full Text PDF PubMed Google Scholar) but does not bind ssDNA when it is covered with the SSB p5 (17Gascón I. Lázaro J.M. Salas M. Nucleic Acids Res. 2000; 28: 2034-2042Crossref PubMed Google Scholar, 23Gutiérrez C. Martı́n G. Sogo J.M. Salas M. J. Biol. Chem. 1991; 266: 2104-2111Abstract Full Text PDF PubMed Google Scholar). In addition, free φ29 DNA polymerase, but not when bound to M13 ssDNA, can degrade a single-stranded oligonucleotide due to its 3′-5′ exonucleolytic activity. M13 DNA that is complexed with an ssDNA-binding protein, therefore, is unable to trap the φ29 DNA polymerase, which can be measured by the 3′-5′ exonucleolytic activity. Thus, φ29 DNA polymerase was added to either naked M13 ssDNA or the M13 ssDNA that was preincubated with increasing amounts of protein p16.7A. Then, 1 min after a 5′ labeled oligonucleotide was added to the mixtures, samples were analyzed for degradation of the oligonucleotide. The assays were carried out in parallel using SSB p5. Fig.5 shows, as expected, that the oligonucleotide was degraded by the φ29 DNA polymerase when the M13 ssDNA trap was omitted (lane 2), but it was not degraded when the reaction mixtures contained naked M13 ssDNA (lane 3). In agreement with previously published results (17Gascón I. Lázaro J.M. Salas M. Nucleic Acids Res. 2000; 28: 2034-2042Crossref PubMed Google Scholar), preincubation of the M13 ssDNA with increasing amounts of SSB p5 resulted in increasing levels of degradation of the oligonucleotide (lanes 4–7). Similar results were obtained when the M13 ssDNA had been preincubated with protein p16.7A (lanes 9–12). The oligonucleotide was not degraded when it was only incubated with the highest concentration of SSB p5 or p16.7A (lanes 8 and 13, respectively). This excludes the possibility that the observed degradation of the oligonucleotide would be the consequence of a contaminant exonuclease in the purified protein preparations. These results, therefore, indicate that protein p16.7A binds to circular M13 ssDNA and that this prevents it from φ29 DNA polymerase binding. Possible helix destabilization activity of protein p16.7A was studied by its ability to displace a 5′-labeled 17-mer oligonucleotide hybridized to its complementary sequence in circular M13 ssDNA molecules. This substrate was incubated without or with increasing amounts of protein p16.7A, after which the samples were analyzed by polyacrylamide gel electrophoresis. The experiments were carried out in parallel using φ29 SSB p5. As shown in Fig. 6, incubation in the absence of protein did not result in release of the labeled oligonucleotide, indicating that the hybrid substrate was stable throughout the experiment. In agreement with previously published results (17Gascón I. Lázaro J.M. Salas M. Nucleic Acids Res. 2000; 28: 2034-2042Crossref PubMed Google Scholar, 24Soengas M.S. Gutiérrez C. Salas M. J. Mol. Biol. 1995; 253: 517-529Crossref PubMed Scopus (66) Google Scholar), the φ29 SSB p5 was able to displace the labeled oligonucleotide from the M13 DNA. However, no displacement of the oligonucleotide was detected when the hybrid substrate was incubated with protein p16.7A up to a concentration of 112 μm. To facilitate the oligonucleotide displacement, these assays were also performed using an oligonucleotide that contained, in addition to the 17 complementary nucleotides, either a non-complementary 5′- (5 thymidines) or a 3′- (5 adenines) tail. Contrary to the SSB p5, protein p16.7A was also unable to displace these oligonucleotides (results not shown). Because protein p16.7A is able to bind M13 ssDNA (see above), these results strongly suggest that protein p16.7A has no helix-destabilizing activity. The φ29-encoded TP and the DNA polymerase are the only two proteins required in a minimal in vitro φ29 DNA replication system (29Blanco L. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6404-6408Crossref PubMed Scopus (75) Google Scholar). Contrary to the φ29 DNA amplification system, the minimal replication system requires high concentrations of template TP-DNA and is limited to one or two rounds of φ29 DNA replication. To study a possible effect of protein p16.7A in this system, in vitroφ29 DNA replication assays were performed in the absence or presence of protein p16.7A using the SSB p5 as a control. The amount of incorporated [α-32P]dAMP was determined (Fig.7 A), after which the samples were subjected to alkaline-agarose gel electrophoresis to determine the size of the synthesized DNA (Fig. 7 B). As reported previously (23Gutie" @default.
W2036768907 created "2016-06-24" @default.
W2036768907 creator A5007474027 @default.
W2036768907 creator A5082925505 @default.
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W2036768907 date "2002-02-01" @default.
W2036768907 modified "2023-09-30" @default.
W2036768907 title "The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein" @default.
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