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. 1995 Aug;177(15):4333–4341. doi: 10.1128/jb.177.15.4333-4341.1995

Domain structure of phage P4 alpha protein deduced by mutational analysis.

G Ziegelin 1, N A Linderoth 1, R Calendar 1, E Lanka 1
PMCID: PMC177181  PMID: 7635818

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.

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Selected References

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