Escherichia coli K-12 has two distinguishable PriA-PriB replication restart pathways.

Abstract

In Escherichia coli, PriA, PriB, PriC, and DnaT proteins mediate three pathways for Replication Restart called PriA-PriB, PriA-PriC, and PriC. PriA is crucial for two of the three pathways. Its absence leads to slow growth, high basal levels of SOS expression, poorly partitioning nucleoids, UV sensitivity, and recombination deficiency. PriA has ATPase and helicase activities and interacts with PriB, DnaT, and single-stranded DNA-binding protein (SSB). priA300 (K230R) and priA301 (C479Y) have no phenotype as single mutants, but each phenocopy a priA-null mutant combined with ∆priB. This suggested that the two priA mutations affected the helicase activity that is required for the PriA-PriC pathway. To further test this, the biochemical activities of purified PriA300 and PriA301 were examined. As expected, PriA300 lacks ATPase and helicase activities but retains the ability to interact with PriB. PriA301, however, retains significant PriB-stimulated helicase activity even though PriA301 interactions with PriB and DNA are weakened. A PriA300,301 variant retains only the ability to interact with DNA in vitro and phenocopies the priA-null phenotype in vivo. This suggests that there are two biochemically and genetically distinct PriA-PriB pathways. One uses PriB-stimulated helicase activity to free a region of ssDNA and the other uses helicase-independent remodeling activity.