The helicase-binding domain of Escherichia coli DnaG primase interacts with the highly conserved C-terminal region of single-stranded DNA-binding protein

Abstract

During bacterial DNA replication, DnaG primase and the χ subunit of DNA polymerase III compete for binding to single-stranded DNA-binding protein (SSB), thus facilitating the switch between priming and elongation. SSB proteins play an essential role in DNA metabolism by protecting single-stranded DNA and by mediating several important protein–protein interactions. Although an interaction of SSB with primase has been previously reported, it was unclear which domains of the two proteins are involved. This study identifies the C-terminal helicase-binding domain of DnaG primase (DnaG-C) and the highly conserved C-terminal region of SSB as interaction sites. By ConSurf analysis, it can be shown that an array of conserved amino acids on DnaG-C forms a hydrophobic pocket surrounded by basic residues, reminiscent of known SSB-binding sites on other proteins. Using protein–protein cross-linking, site-directed mutagenesis, analytical ultracentrifugation and nuclear magnetic resonance spectroscopy, we demonstrate that these conserved amino acid residues are involved in the interaction with SSB. Even though the C-terminal domain of DnaG primase also participates in the interaction with DnaB helicase, the respective binding sites on the surface of DnaG-C do not overlap, as SSB binds to the N-terminal subdomain, whereas DnaB interacts with the ultimate C-terminus.

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

“…Extensions of the amino acid sequence beyond the normal C-terminal phenylalanine have been shown to block SIP interactions 22; 30 and we have shown that the SSB-S1 protein does not interact with χ (Figure S7). We have previously observed inhibition by other SSB derivatives that lack portions of the C-terminal tail.…”

“…27 Furthermore, strand displacement synthesis catalyzed by the Pol III HE in the absence of helicase is dependent on SSB. 28 SSB directly interacts with primase (DnaG) 29; 30 as well as with PriA. 22; 31 This latter interaction is critical to restart of DNA replication at stalled forks and is further enhanced by recruitment of PriB onto DNA.…”

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

“…To this end, we overexpressed and purified RecD2 and SSB and performed an immuno-dot blot to probe for an interaction between RecD2 and SSB. We spotted RecD2, DnaG (a known SSB-interacting protein as a positive control [62]), and BSA (as a negative control) in increasing amounts on a nitrocellulose membrane and incubated the membrane with SSB; SSB was detected using an SSB antiserum as described previously (30,31,35) (Fig. 6A, right).…”

A structural view of bacterial DNA replication

“…Structures of homologs from G. stearothermophilus and Helicobacter pylori have also been reported and show similar topology in spite of poor sequence conservation. Using bioinformatic and site‐directed mutagenesis, the region of the helical bundle that binds to the conserved C‐terminal residues of SSB was identified …”

“…Using bioinformatic and site-directed mutagenesis, the region of the helical bundle that binds to the conserved C-terminal residues of SSB was identified. 27 Helicase DNA helicases, powered by NTP hydrolysis, separate duplex DNA into single strands. The E. coli DNA unwinding helicase, DnaB, belongs to the SF4 family of hexameric helicases that include the replicative helicases of all bacteria as well as bacteriophages T4, T7 and others.…”