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The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein*

  • Alejandro Serna-Rico
    Footnotes
    Affiliations
    Centro de Biologı́a Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain
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  • Margarita Salas
    Correspondence
    To whom correspondence should be addressed: Tel.: 34 91 397 8435; Fax: 34 91 397 8490;
    Affiliations
    Centro de Biologı́a Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain
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  • Wilfried J.J. Meijer
    Footnotes
    Affiliations
    Centro de Biologı́a Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain
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  • Author Footnotes
    * This investigation was supported by National Institutes of Health Research Grant 2R01 GM27242-22, Dirección General de Investigación Cientı́fica y Técnica Grant PB98–0645, European Economic Community Grant ERBFMX CT97 0125, and an Institutional grant from Fundación Ramón Areces.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Holder of a predoctoral fellowship from Gobierno Vasco.
    ¶ Supported by a postdoctoral fellowship from the Spanish Ministry of Education and Culture.
Open AccessPublished:December 10, 2001DOI:https://doi.org/10.1074/jbc.M109312200
      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.
      • Kornberg A.
      • Baker T.A.
      ). 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 (
      • Meijer W.J.J.
      • Lewis P.J.
      • Errington J.
      • Salas M.
      ). The detailed knowledge of its in vitro mechanism of DNA replication (for reviews, see Refs.
      • Salas M.
      • Rojo F.
      and
      • Salas M.
      • Miller J.T.
      • Leis J.
      • DePamphilis M.L.
      ) made φ29 an attractive system for this study.
      The genome of φ29 is a linear double-stranded DNA (dsDNA)
      dsDNA
      double-stranded DNA
      ssDNA
      single-stranded DNA
      TP
      terminal protein
      SSB
      single-stranded DNA-binding protein
      1dsDNA
      double-stranded DNA
      ssDNA
      single-stranded DNA
      TP
      terminal protein
      SSB
      single-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.
      • Salas M.
      • Rojo F.
      and
      • Rojo F.
      • Mencı́a M.
      • Monsalve M.
      • Salas M.
      ). 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.
      Figure thumbnail gr1
      Figure 1Schematic representation of the genetic and transcriptional organization of the φ29 genome and its in vitro DNA replication mechanism. A, genetic and transcription map of phage φ29 DNA. The direction of transcription and length of the transcripts are indicated by arrows. Positions of the various genes, indicated withnumbers, are shown between the two DNA strands. The proteins relevant for this work are indicated. The positions of the open reading frames 16.9, 16.8, 16.6, and 16.5, located at the right side of the φ29 genome, are indicated with the numbers .9,.8, .6, and .5, respectively. TD1, position of a bidirectional transcriptional terminator. Filled circles represent the covalently linked φ29 terminal protein. The map is adapted from Mellado et al. (
      • Mellado R.P.
      • Moreno F.
      • Viñuela E.
      • Salas M.
      • Reilly B.E.
      • Anderson D.L.
      ). DBP, dsDNA-binding protein. B, Mechanism of in vitroφ29 DNA replication. See the introduction for details.Circles and triangles represent TP and DNA polymerase, respectively. Synthesized DNA strands are indicated with broken lines. Adapted from Meijer et al. (
      • Meijer W.J.J.
      • Lewis P.J.
      • Errington J.
      • Salas M.
      ).
      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.
      • Salas M.
      • Rojo F.
      ,
      • Salas M.
      • Miller J.T.
      • Leis J.
      • DePamphilis M.L.
      , and
      • Meijer W.J.J.
      • Horcajadas J.A.
      • Salas M.
      ). 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.
      • Firshein W.
      ). Also, replication of φ29 DNA takes place at the membrane of the infected cell (
      • Meijer W.J.J.
      • Lewis P.J.
      • Errington J.
      • Salas M.
      ,
      • Ivarie R.D.
      • Pène J.J.
      ,
      • Bravo A.
      • Salas M.
      ). 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 (
      • Meijer W.J.J.
      • Serna-Rico A.
      • Salas M.
      ). 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.

      Acknowledgments

      We thank L. Blanco for critical reading of the manuscript, Marı́a Teresa Rejas for electron microscopy assistance, and L. Villar and J. M. Lázaro for protein purification.

      REFERENCES

        • Kornberg A.
        • Baker T.A.
        DNA Replication. W. H. Freeman and Co., San Fransisco1992
        • Meijer W.J.J.
        • Lewis P.J.
        • Errington J.
        • Salas M.
        EMBO J. 2000; 19: 4182-4190
        • Salas 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: 843-858
        • Salas 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: 131-176
        • Rojo F.
        • Mencı́a M.
        • Monsalve M.
        • Salas M.
        Prog. Nucleic Acid Res. Mol. Biol. 1998; 60: 29-46
        • Meijer W.J.J.
        • Horcajadas J.A.
        • Salas M.
        Microbiol. Mol. Biol. Rev. 2001; 65: 261-287
        • Firshein W.
        Annu. Rev. Microbiol. 1989; 43: 89-120
        • Ivarie R.D.
        • Pène J.J.
        Virology. 1973; 52: 351-362
        • Bravo A.
        • Salas M.
        J. Mol. Biol. 1997; 269: 102-112
        • Meijer W.J.J.
        • Serna-Rico A.
        • Salas M.
        Mol. Microbiol. 2001; 39: 731-746
        • Moreno F.
        • Camacho A.
        • Viñuela E.
        • Salas M.
        Virology. 1974; 62: 1-16
        • Jiménez F.
        • Camacho A.
        • de la Torre J.
        • Viñuela E.
        • Salas M.
        Eur. J. Biochem. 1977; 73: 57-72
        • Sambrook J.
        • Fritsch E.F.
        • Maniatis T.
        Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989
        • Serna-Rico A.
        • Illana B.
        • Salas M.
        • Meijer W.J.J.
        J. Biol. Chem. 2000; 275: 40529-40538
        • Sogo J.M.
        • Thoma F.
        Methods Enzymol. 1989; 170: 142-165
        • Sogo 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: 61-79
        • Gascón I.
        • Lázaro J.M.
        • Salas M.
        Nucleic Acids Res. 2000; 28: 2034-2042
        • Blanco L.
        • Bernad A.
        • Esteban J.A.
        • Salas M.
        J. Biol. Chem. 1992; 267: 1225-1230
        • Blanco L.
        • Lázaro J.M.
        • de Vega M.
        • Bonnin A.
        • Salas M.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12198-12202
        • Esteban J.A.
        • Blanco L.
        • Villar L.
        • Salas M.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2921-2926
        • Wollenzien P.L.
        Gasparro F.P. Psoralen-DNA Photobiology. CRC Press, Inc., Boca Raton, FL1988: 51-85
        • Gutiérrez C.
        • Sogo J.M.
        • Salas M.
        J. Mol. Biol. 1991; 222: 983-994
        • Gutiérrez C.
        • Martı́n G.
        • Sogo J.M.
        • Salas M.
        J. Biol. Chem. 1991; 266: 2104-2111
        • Soengas M.S.
        • Gutiérrez C.
        • Salas M.
        J. Mol. Biol. 1995; 253: 517-529
        • Martı́n G.
        • Lázaro J.M.
        • Méndez E.
        • Salas M.
        Nucleic Acids Res. 1989; 17: 3663-3672
        • Soengas M.S.
        • Esteban J.A.
        • Salas M.
        • Gutiérrez C.
        J. Mol. Biol. 1994; 239: 213-226
        • Gascón I.
        • Gutiérrez C.
        • Salas M.
        J. Mol. Biol. 2000; 296: 989-999
        • Blanco L.
        • Bernad A.
        • Lázaro J.M.
        • Martı́n G.
        • Garmendia C.
        • Salas M.
        J. Biol. Chem. 1989; 264: 8935-8940
        • Blanco L.
        • Salas M.
        Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6404-6408
        • Meyer R.R.
        • Laine P.S.
        Microbiol. Rev. 1990; 54: 342-380
        • Chase J.W.
        • Williams K.R.
        Annu. Rev. Biochem. 1986; 55: 103-136
        • Kelman Z.
        • O'Donnell M.
        Curr. Opin. Genet. Dev. 1994; 4: 185-195
        • Stillman B.
        Cell. 1994; 78: 725-728
        • Salas M.
        Annu. Rev. Biochem. 1991; 60: 39-71
        • Salas M.
        • Freire R.
        • Soengas M.S.
        • Esteban J.A.
        • Méndez J.
        • Bravo A.
        • Serrano M.
        • Blasco M.A.
        • Lázaro J.M.
        • Blanco L.
        • Gutiérrez C.
        • Hermoso J.M.
        FEMS Microbiol. Rev. 1995; 17: 73-82
        • Mellado R.P.
        • Peñalva M.A.
        • Inciarte M.R.
        • Salas M.
        Virology. 1980; 104: 84-96
        • Mellado R.P.
        • Moreno F.
        • Viñuela E.
        • Salas M.
        • Reilly B.E.
        • Anderson D.L.
        J. Virol. 1976; 19: 495-500