Phage φ29 Terminal Protein Residues Asn80 and Tyr82 Are Recognition Elements of the Replication Origins

Abstract

Initiation of phage φ29 DNA replication starts with the recognition of the origin of replication, located at both ends of the linear DNA, by a heterodimer formed by the φ29 terminal protein (TP) and the φ29 DNA polymerase. The parental TP, covalently linked to the DNA ends, is one of the main components of the replication origin. Here we provide evidence that recognition of the origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on amino acid sequence comparisons, various φ29 TP mutants were generated at conserved amino acid residues from positions 61 to 87.In vitro φ29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for functional interaction between primer and parental TP required for recognition of the origin of replication. Although these mutant TPs can form functional heterodimers with φ29 DNA polymerase that are able to recognize the origin of replication, these heterodimers are not able to recognize an origin containing a mutant TP. Initiation of phage φ29 DNA replication starts with the recognition of the origin of replication, located at both ends of the linear DNA, by a heterodimer formed by the φ29 terminal protein (TP) and the φ29 DNA polymerase. The parental TP, covalently linked to the DNA ends, is one of the main components of the replication origin. Here we provide evidence that recognition of the origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on amino acid sequence comparisons, various φ29 TP mutants were generated at conserved amino acid residues from positions 61 to 87.In vitro φ29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for functional interaction between primer and parental TP required for recognition of the origin of replication. Although these mutant TPs can form functional heterodimers with φ29 DNA polymerase that are able to recognize the origin of replication, these heterodimers are not able to recognize an origin containing a mutant TP. DNA replication is a semiconservative process in which a DNA polymerase uses one DNA strand as a template for the synthesis of its complementary strand. All DNA polymerases require a preexisting primer to initiate DNA synthesis (1Kornberg A. Baker A. DNA Replication. Freeman, San Francisco1992: 103-106Google Scholar). In many cases, the primer is a short RNA or DNA molecule. In linear DNAs, which are unable to form circular or hairpin structures during replication, replication of the genome ends cannot take place via RNA or DNA priming. Several mechanisms have evolved to solve the problem of initiation of DNA replication in such linear genomes (2Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 131-176Google Scholar). In the case of viral linear genomes, the OH group of a serine, threonine, or tyrosine residue of a terminal protein (TP) 1The abbreviations used are: TP, terminal protein. 1The abbreviations used are: TP, terminal protein. molecule serves as a primer for DNA replication, and as a consequence, the TP molecule becomes covalently attached to the 5′-terminal nucleotide. This mechanism of initiation of replication is called protein priming (reviewed in Ref. 3Salas M. Annu. Rev. Biochem. 1991; 60: 39-71Crossref PubMed Scopus (344) Google Scholar). Once bound to the DNA, the TP may serve additional functions such as assisting in DNA packaging (4Bjornsti M.A. Reilly B.E. Anderson D.L. J. Virol. 1982; 41: 508-517Crossref PubMed Google Scholar, 5Grimes S. Anderson D. J. Mol. Biol. 1989; 209: 101-108Crossref PubMed Scopus (20) Google Scholar), protection against nucleases (6Dunsworth-Browne M. Schell R.E. Berk A.J. Nucleic Acids Res. 1980; 8: 543-554Crossref PubMed Scopus (19) Google Scholar), enhancement of infectivity (7Hirokawa H. Proc. Natl. Acad. Sci. U . S . A. 1972; 69: 1555-1559Crossref PubMed Scopus (57) Google Scholar, 8Ronda C. López R. Gómez A. Garcı́a E. J. Virol. 1983; 48: 721-730Crossref PubMed Google Scholar), stimulation of template activity (9Pronk R. Stuvier M.H. van der Vliet P.C. Chromosoma (Berl.). 1992; 102: 39-45Crossref Scopus (18) Google Scholar), matrix attachment (10Schaack J. Yew-Wai Ho J. Freimuth P. Shenk T. Gene (Amst.). 1990; 4: 1197-1208Google Scholar), stimulation of transcription (10Schaack J. Yew-Wai Ho J. Freimuth P. Shenk T. Gene (Amst.). 1990; 4: 1197-1208Google Scholar), and recruitment of a new TP to the origin (11Gutiérrez J. Vinós J. Prieto I. Méndez E. Hermoso J.M. Salas M. Virology. 1986; 155: 474-483Crossref PubMed Scopus (25) Google Scholar). As a step previous to the initiation of replication, TP interacts with the DNA polymerase to form a heterodimer (12Blanco L. Prieto I. Gutiérrez J. Bernad A. Lázaro J.M. Hermoso J.M. Salas M. J. Virol. 1987; 61: 3983-3991Crossref PubMed Google Scholar). To distinguish between the different functions of TP, we will refer to the TP bound to the DNA ends as parental TP and the TP in the heterodimer with the DNA polymerase as primer TP. Interaction of primer TP and/or DNA polymerase with parental TP may increase the affinity of the TP/DNA polymerase heterodimer for the origin or could assist the latter for its correct positioning at the origin, which may be important for initiation of DNA replication at the correct position.The protein-priming mechanism of DNA replication has been mainly studied in the Bacillus subtilis bacteriophage φ29 and adenoviruses (see Refs. 2Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 131-176Google Scholar, 3Salas M. Annu. Rev. Biochem. 1991; 60: 39-71Crossref PubMed Scopus (344) Google Scholar, 13Hay R.T. DePamphilis M.L. Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 699-719Google Scholar, and 14van der Vliet P.C. DePamphilis M.L. Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 87-118Google Scholar for review). The φ29 genome is a 19,285-base pair linear double-stranded DNA molecule (15Vlcek C. Paces V. Gene (Amst.). 1986; 46: 215-225Crossref PubMed Scopus (57) Google Scholar) with a phage-encoded 31-kDa TP covalently attached to the 5′ termini (16Salas M. Mellado R.P. Viñuela E. Sogo J.M. J. Mol. Biol. 1978; 119: 269-291Crossref PubMed Scopus (111) Google Scholar). Genetic studies and the development of an in vitro DNA replication system (17Peñalva M.A. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1982; 79: 5522-5526Crossref PubMed Scopus (115) Google Scholar) led to the identification of the origins of replication at each end of the DNA molecule (18Inciarte M.R. Salas M. Sogo J.M. J. Virol. 1980; 34: 187-199Crossref PubMed Google Scholar, 19Sogo J.M. Garcı́a J.A. Peñalva M.A. Salas M. J. Virol. 1982; 116: 1-18Crossref Scopus (28) Google Scholar). To activate the initiation of replication, dsDNA-binding protein p6 forms a nucleoprotein complex that would help to open the DNA ends (20Serrano M. Salas M. Hermoso J.M. Science. 1990; 248: 1012-1016Crossref PubMed Scopus (64) Google Scholar). A primer TP/DNA polymerase heterodimer recognizes the origin of replication, probably through protein-protein interaction of the primer TP and/or φ29 DNA polymerase with the parental TP. Then, the φ29 DNA polymerase catalyzes the addition of the first dAMP (21Blanco L. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1984; 81: 5325-5329Crossref PubMed Scopus (101) Google Scholar) to the OH group provided by Ser232 of the primer TP (22Hermoso J.M. Méndez E. Soriano F. Salas M. Nucleic Acids Res. 1985; 13: 7715-7728Crossref PubMed Scopus (71) Google Scholar). The formation of this first TP-dAMP is directed by the second nucleotide at the 3′-end of the template and then slides back one nucleotide to recover the terminal nucleotide (23Méndez J. Blanco L. Esteban J.A. Bernad A. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1992; 89: 9579-9583Crossref PubMed Scopus (98) Google Scholar). Following initiation, the same φ29 DNA polymerase molecule completes replication of the parental strand.φ29 parental TP is an important requirement for in vitroinitiation of φ29 DNA replication (24Garcı́a J.A. Peñalva M.A. Blanco L. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1984; 81: 80-84Crossref PubMed Scopus (21) Google Scholar). Two lines of evidence show that parental TP is also required in vivo. First, in a mixed infection experiment at 42 °C using phages that have a thermosensitive (ts) mutation in either gene 2 (encoding the DNA polymerase) or gene 3 (encoding TP), most of the phage progeny had the ts2 genotype (16Salas M. Mellado R.P. Viñuela E. Sogo J.M. J. Mol. Biol. 1978; 119: 269-291Crossref PubMed Scopus (111) Google Scholar), and second, successful transfection required an intact gene 3 product (17Peñalva M.A. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1982; 79: 5522-5526Crossref PubMed Scopus (115) Google Scholar, 25Salas M. Garcı́a J.A. Peñalva M.A. Blanco L. Prieto I. Mellado R.P. Lázaro J.M. Pastrana R. Escarmı́s C. Hermoso J.M. Cozzarelli N.R. Mechanisms of DNA Replication and Recombination. Alan R. Liss Inc., New York1983: 203-223Google Scholar). Moreover, formation of the TP-dAMP initiation complex was obtained using as template the TP-DNA isolated from the closely related Bacillus phage ø15 but not from the more distantly related Bacillus phage GA-1 or the pneumococcal phage Cp-1. The lack of activity in the initiation reaction of the TP-DNA isolated from these latter two phages could be because of differences in the parental TPs (24Garcı́a J.A. Peñalva M.A. Blanco L. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1984; 81: 80-84Crossref PubMed Scopus (21) Google Scholar).Structure-function studies and biochemical characterization of φ29 TP provide a basis to gain insight about the different roles of this protein in φ29 DNA replication. As shown here, a mutational analysis of φ29 TP at conserved amino acids from positions 61 to 87 revealed that residues Asn80 and Tyr82 are important for recognition of the origin of replication. The results obtained indicate that parental TP plays an important role as a structural part of the replication origin through functional interactions with the primer TP. DNA replication is a semiconservative process in which a DNA polymerase uses one DNA strand as a template for the synthesis of its complementary strand. All DNA polymerases require a preexisting primer to initiate DNA synthesis (1Kornberg A. Baker A. DNA Replication. Freeman, San Francisco1992: 103-106Google Scholar). In many cases, the primer is a short RNA or DNA molecule. In linear DNAs, which are unable to form circular or hairpin structures during replication, replication of the genome ends cannot take place via RNA or DNA priming. Several mechanisms have evolved to solve the problem of initiation of DNA replication in such linear genomes (2Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 131-176Google Scholar). In the case of viral linear genomes, the OH group of a serine, threonine, or tyrosine residue of a terminal protein (TP) 1The abbreviations used are: TP, terminal protein. 1The abbreviations used are: TP, terminal protein. molecule serves as a primer for DNA replication, and as a consequence, the TP molecule becomes covalently attached to the 5′-terminal nucleotide. This mechanism of initiation of replication is called protein priming (reviewed in Ref. 3Salas M. Annu. Rev. Biochem. 1991; 60: 39-71Crossref PubMed Scopus (344) Google Scholar). Once bound to the DNA, the TP may serve additional functions such as assisting in DNA packaging (4Bjornsti M.A. Reilly B.E. Anderson D.L. J. Virol. 1982; 41: 508-517Crossref PubMed Google Scholar, 5Grimes S. Anderson D. J. Mol. Biol. 1989; 209: 101-108Crossref PubMed Scopus (20) Google Scholar), protection against nucleases (6Dunsworth-Browne M. Schell R.E. Berk A.J. Nucleic Acids Res. 1980; 8: 543-554Crossref PubMed Scopus (19) Google Scholar), enhancement of infectivity (7Hirokawa H. Proc. Natl. Acad. Sci. U . S . A. 1972; 69: 1555-1559Crossref PubMed Scopus (57) Google Scholar, 8Ronda C. López R. Gómez A. Garcı́a E. J. Virol. 1983; 48: 721-730Crossref PubMed Google Scholar), stimulation of template activity (9Pronk R. Stuvier M.H. van der Vliet P.C. Chromosoma (Berl.). 1992; 102: 39-45Crossref Scopus (18) Google Scholar), matrix attachment (10Schaack J. Yew-Wai Ho J. Freimuth P. Shenk T. Gene (Amst.). 1990; 4: 1197-1208Google Scholar), stimulation of transcription (10Schaack J. Yew-Wai Ho J. Freimuth P. Shenk T. Gene (Amst.). 1990; 4: 1197-1208Google Scholar), and recruitment of a new TP to the origin (11Gutiérrez J. Vinós J. Prieto I. Méndez E. Hermoso J.M. Salas M. Virology. 1986; 155: 474-483Crossref PubMed Scopus (25) Google Scholar). As a step previous to the initiation of replication, TP interacts with the DNA polymerase to form a heterodimer (12Blanco L. Prieto I. Gutiérrez J. Bernad A. Lázaro J.M. Hermoso J.M. Salas M. J. Virol. 1987; 61: 3983-3991Crossref PubMed Google Scholar). To distinguish between the different functions of TP, we will refer to the TP bound to the DNA ends as parental TP and the TP in the heterodimer with the DNA polymerase as primer TP. Interaction of primer TP and/or DNA polymerase with parental TP may increase the affinity of the TP/DNA polymerase heterodimer for the origin or could assist the latter for its correct positioning at the origin, which may be important for initiation of DNA replication at the correct position. The protein-priming mechanism of DNA replication has been mainly studied in the Bacillus subtilis bacteriophage φ29 and adenoviruses (see Refs. 2Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 131-176Google Scholar, 3Salas M. Annu. Rev. Biochem. 1991; 60: 39-71Crossref PubMed Scopus (344) Google Scholar, 13Hay R.T. DePamphilis M.L. Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 699-719Google Scholar, and 14van der Vliet P.C. DePamphilis M.L. Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 87-118Google Scholar for review). The φ29 genome is a 19,285-base pair linear double-stranded DNA molecule (15Vlcek C. Paces V. Gene (Amst.). 1986; 46: 215-225Crossref PubMed Scopus (57) Google Scholar) with a phage-encoded 31-kDa TP covalently attached to the 5′ termini (16Salas M. Mellado R.P. Viñuela E. Sogo J.M. J. Mol. Biol. 1978; 119: 269-291Crossref PubMed Scopus (111) Google Scholar). Genetic studies and the development of an in vitro DNA replication system (17Peñalva M.A. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1982; 79: 5522-5526Crossref PubMed Scopus (115) Google Scholar) led to the identification of the origins of replication at each end of the DNA molecule (18Inciarte M.R. Salas M. Sogo J.M. J. Virol. 1980; 34: 187-199Crossref PubMed Google Scholar, 19Sogo J.M. Garcı́a J.A. Peñalva M.A. Salas M. J. Virol. 1982; 116: 1-18Crossref Scopus (28) Google Scholar). To activate the initiation of replication, dsDNA-binding protein p6 forms a nucleoprotein complex that would help to open the DNA ends (20Serrano M. Salas M. Hermoso J.M. Science. 1990; 248: 1012-1016Crossref PubMed Scopus (64) Google Scholar). A primer TP/DNA polymerase heterodimer recognizes the origin of replication, probably through protein-protein interaction of the primer TP and/or φ29 DNA polymerase with the parental TP. Then, the φ29 DNA polymerase catalyzes the addition of the first dAMP (21Blanco L. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1984; 81: 5325-5329Crossref PubMed Scopus (101) Google Scholar) to the OH group provided by Ser232 of the primer TP (22Hermoso J.M. Méndez E. Soriano F. Salas M. Nucleic Acids Res. 1985; 13: 7715-7728Crossref PubMed Scopus (71) Google Scholar). The formation of this first TP-dAMP is directed by the second nucleotide at the 3′-end of the template and then slides back one nucleotide to recover the terminal nucleotide (23Méndez J. Blanco L. Esteban J.A. Bernad A. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1992; 89: 9579-9583Crossref PubMed Scopus (98) Google Scholar). Following initiation, the same φ29 DNA polymerase molecule completes replication of the parental strand. φ29 parental TP is an important requirement for in vitroinitiation of φ29 DNA replication (24Garcı́a J.A. Peñalva M.A. Blanco L. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1984; 81: 80-84Crossref PubMed Scopus (21) Google Scholar). Two lines of evidence show that parental TP is also required in vivo. First, in a mixed infection experiment at 42 °C using phages that have a thermosensitive (ts) mutation in either gene 2 (encoding the DNA polymerase) or gene 3 (encoding TP), most of the phage progeny had the ts2 genotype (16Salas M. Mellado R.P. Viñuela E. Sogo J.M. J. Mol. Biol. 1978; 119: 269-291Crossref PubMed Scopus (111) Google Scholar), and second, successful transfection required an intact gene 3 product (17Peñalva M.A. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1982; 79: 5522-5526Crossref PubMed Scopus (115) Google Scholar, 25Salas M. Garcı́a J.A. Peñalva M.A. Blanco L. Prieto I. Mellado R.P. Lázaro J.M. Pastrana R. Escarmı́s C. Hermoso J.M. Cozzarelli N.R. Mechanisms of DNA Replication and Recombination. Alan R. Liss Inc., New York1983: 203-223Google Scholar). Moreover, formation of the TP-dAMP initiation complex was obtained using as template the TP-DNA isolated from the closely related Bacillus phage ø15 but not from the more distantly related Bacillus phage GA-1 or the pneumococcal phage Cp-1. The lack of activity in the initiation reaction of the TP-DNA isolated from these latter two phages could be because of differences in the parental TPs (24Garcı́a J.A. Peñalva M.A. Blanco L. Salas M. Proc. Natl. Acad. Sci. U . S . A. 1984; 81: 80-84Crossref PubMed Scopus (21) Google Scholar). Structure-function studies and biochemical characterization of φ29 TP provide a basis to gain insight about the different roles of this protein in φ29 DNA replication. As shown here, a mutational analysis of φ29 TP at conserved amino acids from positions 61 to 87 revealed that residues Asn80 and Tyr82 are important for recognition of the origin of replication. The results obtained indicate that parental TP plays an important role as a structural part of the replication origin through functional interactions with the primer TP. We are very grateful to L. Villar for help during purification of proteins and to Dr. Maite Rejas for the electron microscopy work.