Conserved residues of the C‐terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome

Abstract

SummaryThe bacterial primosome comprises the replicative homo-hexameric ring helicase DnaB and the primase DnaG. It is an integral component of the replisome as it unwinds the parental DNA duplex to allow progression of the replication fork, synthesizes the initiation primers at the replication origin, oriC, and the primers required for Okazaki fragment synthesis during lagging strand replication. The interaction between the two component proteins is mediated by a distinct C-terminal domain (p16) of the primase. Both proteins mutually regulate each other's activities and a putative network of conserved residues has been proposed to mediate these effects. We have targeted 10 residues from this network. To investigate the functional contributions of these residues to the primase, ATPase and helicase activities of the primosome, we have used site-directed mutagenesis and in vitro functional assays. Five of these residues (E464, H494, R495, Y548 and R555) exhibited some functional significance while the remaining five (E483, R484, E506, D512 and E530) exhibited no effects. E464 participates in functional modulation of the primase activity, whereas H494, R495 and R555 participate in allosteric functional modulation of the ATPase and/or helicase activities. Y548 contributes directly to the structural interaction with DnaB.

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

“…2C). The stimulatory effect of ssDNA on ATPase activity of DnaB was in consistent with that in enzymecoupled assays [14,26], however, the assay here was much easier. Interestingly, high concentration of DnaI Bsu (DnaI and DnaB with the molar ratio higher than 3:1, DnaB referred to was monomer here) showed inhibition on the ATPase activity of DnaB, the increasing of the DnaI concentration resulted in further inhibition of ATPase activity (Fig.…”

Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp

“…During this step, DnaG binds to the replicative helicase DnaB, forming a complex known as the primosome. This interaction formed by DnaB and DnaG has been shown to modulate the activities of both enzymes (39).…”

An in Trans Interaction at the Interface of the Helicase and Primase Domains of the Hexameric Gene 4 Protein of Bacteriophage T7 Modulates Their Activities

“…1A) (27)(28)(29)(30)(31). The association of DnaB with DnaG alters sequence recognition by DnaG and affects the length of primers synthesized (28,(31)(32)(33). Furthermore, cooperative binding of two or three DnaG monomers to the hexameric DnaB can halt translocation of DnaB on DNA (34).…”

“…In bacteria such as E. coli, Bacillus stearothermophilus, and Staphylococcus aureus, this interaction is mediated through two structurally similar regions: the helicase-binding domain (p16 domain) located at the C terminus of the DnaG primase and the p17 domain of the DnaB helicase located at the N terminus of the protein (see Fig. 1A) (27)(28)(29)(30)(31). The association of DnaB with DnaG alters sequence recognition by DnaG and affects the length of primers synthesized (28,(31)(32)(33).…”

“…Such "association and dissociation" between the helicase and primase mediated by the p16 and p17 domains are believed to coordinate DNA synthesis by regulating the initiation of Okazaki fragment synthesis (6,35,36). Mutations in the p16 domain of DnaG can either affect the ability of the two proteins to form a complex, enhance the primase activity, or modulate the ATPase and/or helicase activities allosterically (31).…”