PMID:10572298
| Citation |
Masai, H, Deneke, J, Furui, Y, Tanaka, T and Arai, KI Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: involvement of ATPase/helicase activity of PriA for inducible stable DNA replication. Biochimie 81:847-57 |
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| Abstract |
The E. coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the phiX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. B. subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its biochemical properties. BsPriA binds specifically to both n'-pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'-pas albeit with a specific activity about 30% of that of E. coli PriA. However, it is not capable of supporting n'-pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the phiX174-type primosome. We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance. Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant. K230D was previously reported to be fully functional in assembly of the phiX174-type primosome at a single-stranded n'-pas. Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo. |
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| Keywords |
Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Amino Acid Sequence; Bacillus subtilis/genetics; Bacillus subtilis/metabolism; Bacteriophage phi X 174/genetics; Bacteriophage phi X 174/metabolism; Base Sequence; Cloning, Molecular; DNA Helicases/genetics; DNA Helicases/metabolism; DNA Primers/genetics; DNA Replication; DNA, Bacterial/biosynthesis; DNA, Bacterial/genetics; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli/metabolism; Escherichia coli Proteins; Genes, Bacterial; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids/genetics; Radiation Tolerance/genetics; Recombination, Genetic; Transformation, Genetic; Ultraviolet Rays |
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