Biochemical analysis of PriA Helicases from Gram‐positive Bacteria involved in DNA Replication Restart

Abstract

DNA replication forks often encounter template DNA lesions, which can stall progression of the replication forks. In gram‐positive bacteria, the PriA‐dependent pathway is the major replication restart mechanism and it requires several primosome proteins. Among them, the PriA protein—a 3′ to 5′ superfamily‐2 DNA helicase—is the key factor recognizing DNA lesions and it further recruits other proteins. Here, we investigated the PriA ATPase and helicase activities of Streptococcus pneumoniae (SpPriA) and Geobacillus stearothermophilus (GstPriA) through biochemical and kinetic analyses. Using various DNA duplexes with unstructured single‐stranded regions, we observed distinct unwinding abilities for GstPriA and SpPriA according to increased GC content of duplex DNA substrate. In contrast to the unwinding activity of GstPriA helicase, we found that SpPriA is unable to unwind duplex DNA substrate with high GC content. However, the presence of DnaD loader protein allows SpPriA to unwind such duplex DNA. Our findings suggest that SpPriA possesses limited unwinding ability in high GC DNA and that DnaD acts as an accessory protein to destabilize stalled replication forks through its single‐stranded DNA binding ability, thereby enhancing PriA unwinding activity.