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|Título :||Phage φ29 Terminal Protein Residues Asn80 and Tyr82 Are Recognition Elements of the Replication Origins|
|Autor :||Illana, Belén, Lázaro, José M., Gutiérrez, C., Meijer, Wilhelmus J. J., Blanco, Luis, Salas, Margarita|
|Fecha de publicación :||21-May-1999|
|Editor:||American Society for Biochemistry and Molecular Biology|
|Resumen:||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 (1). 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 (2). In the case of viral linear genomes, the OH group of a serine, threonine, or tyrosine residue of a terminal protein (TP)1 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. 3). Once bound to the DNA, the TP may serve additional functions such as assisting in DNA packaging (4, 5), protection against nucleases (6), enhancement of infectivity (7, 8), stimulation of template activity (9), matrix attachment (10), stimulation of transcription (10), and recruitment of a new TP to the origin (11). As a step previous to the initiation of replication, TP interacts with the DNA polymerase to form a heterodimer (12). 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. 2, 3, 13, and 14 for review). The φ29 genome is a 19,285-base pair linear double-stranded DNA molecule (15) with a phage-encoded 31-kDa TP covalently attached to the 5′ termini (16). Genetic studies and the development of an in vitro DNA replication system (17) led to the identification of the origins of replication at each end of the DNA molecule (18, 19). To activate the initiation of replication, dsDNA-binding protein p6 forms a nucleoprotein complex that would help to open the DNA ends (20). 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 (21) to the OH group provided by Ser232 of the primer TP (22). 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 (23). 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 (24). 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 (16), and second, successful transfection required an intact gene 3 product (17, 25). 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 (24).
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.
|Versión del editor:||http://dx.doi.org/10.1074/jbc.274.21.15073|
|Citación :||Journal of Biological Chemistry 274: 15073-15079 (1999)|
|Appears in Collections:||(CBM) Artículos|
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