Dissecting the role of the ϕ29 terminal protein DNA binding residues in viral DNA replication.
Phage ϕ29 DNA replication takes place by a protein-priming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the ϕ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the Km for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the ϕ29 double-stranded DNA binding protein p6 in the reaction, which decreased the Km of the DNA polymerase for dATP about 130-190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification.
Instituto de Biología Molecular 'Eladio Viñuela' (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Isabel
I
Holguera
Instituto de Biología Molecular 'Eladio Viñuela' (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain dmunoz@cnio.es.
Daniel
D
Muñoz-Espín
Instituto de Biología Molecular 'Eladio Viñuela' (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular 'Severo Ochoa' (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain msalas@cbm.csic.es.
Margarita
M
Salas
D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation; D011485:Protein Binding; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C413452:phage phi29 protein p6; C040784:terminal protein, Bacillus phage phi29; D004259:DNA-Directed DNA Polymerase