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

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 phi X174-type primosome and plays critical roles in RecA-dependent ind ucible and constitutive stable DNA replication (iSDR and cSDR, respectively ) as well as in recombination-dependent repair of double-stranded DNA break s. B. subtilis PriA (BsPriA) protein contains the conserved helicase domain s as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its bio chemical properties. BsPriA binds specifically to both n'-pas (primosome as sembly 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 phi X174-type primosome. We also show that a zinc finger mu tant is not able to support recombination-dependent DNA replication, as mea sured by the level of iSDR after a period of thymine starvation, nor wild-t ype level of growth, cell morphology and UV resistance. Unexpectedly, we di scovered that an ATPase-deficient mutant (K230D) is not able to support iSD R to a full extent, although it can restore normal growth rate and UV resis tance as well as non-filamentous morphology in priAl::kan mutant. K230D was previously reported to be fully functional in assembly of the phi X174-typ e primosome at a single-stranded n'-pas. Our results indicate that ATP hydr olysis/helicase activity of PriA may be specifically required for DNA repli cation from recombination intermediates in vivo. (C) 1999 Societe francaise de biochimie et biologie moleculaire/Editions scientifiques et medicales E lsevier SAS.