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 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.
Masai
H
H
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Deneke
J
J
Furui
Y
Y
Tanaka
T
T
Arai
K I
KI
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D010584:Bacteriophage phi X 174 / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011836:Radiation Tolerance / Q000235:genetics; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D014466:Ultraviolet Rays
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D000251:Adenosine Triphosphatases; C506743:priA protein, E coli; D004265:DNA Helicases