title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35.
abstract: Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in a number of linear genomes of viruses, linear plasmids and mobile elements. By this mechanism, a so-called terminal protein (TP) primes replication and becomes covalently linked to the genome ends. Bam35 belongs to a group of temperate tectiviruses infecting Gram-positive bacteria, predicted to replicate their genomes by a protein-primed mechanism. Here, we characterize Bam35 replication as an alternative model of protein-priming DNA replication. First, we analyze the role of the protein encoded by the ORF4 as the TP and characterize the replication mechanism of the viral genome (TP-DNA). Indeed, full-length Bam35 TP-DNA can be replicated using only the viral TP and DNA polymerase. We also show that DNA replication priming entails the TP deoxythymidylation at conserved tyrosine 194 and that this reaction is directed by the third base of the template strand. We have also identified the TP tyrosine 172 as an essential residue for the interaction with the viral DNA polymerase. Furthermore, the genetic information of the first nucleotides of the genome can be recovered by a novel single-nucleotide jumping-back mechanism. Given the similarities between genome inverted terminal repeats and the genes encoding the replication proteins, we propose that related tectivirus genomes can be replicated by a similar mechanism.
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Nicolás Cabrera, 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Berjón-Otero
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Nicolás Cabrera, 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Laurentino
authors.initials: L
authors.lastname: Villar
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Nicolás Cabrera, 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain msalas@cbm.csic.es.
authors.forename: Margarita
authors.initials: M
authors.lastname: Salas
authors.affiliation: Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Nicolás Cabrera, 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain mredrejo@cbm.csic.es.
authors.forename: Modesto
authors.initials: M
authors.lastname: Redrejo-Rodríguez
mesh_terms: D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004279:DNA, Viral; D016679:Genome, Viral; D016366:Open Reading Frames / Q000235:genetics; D011485:Protein Binding; D017916:Tectiviridae / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins