title: Domain structure of phage P4 alpha protein deduced by mutational analysis.
abstract: Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.
authors.affiliation: Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Ziegelin
authors.forename: N A
authors.initials: NA
authors.lastname: Linderoth
authors.forename: R
authors.initials: R
authors.lastname: Calendar
authors.forename: E
authors.initials: E
authors.lastname: Lanka
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics*; D004265:DNA Helicases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017900:Myoviridae / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D018741:Replication Origin; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C095083:alpha protein, Bacteriophage P4; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases