Domain structure of phage P4 alpha protein deduced by mutational analysis.
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.
Ziegelin
G
G
Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.
Linderoth
N A
NA
Calendar
R
R
Lanka
E
E
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*
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