A Central Role for SSB in Escherichia coli RecQ DNA Helicase Function

Abstract

RecQ DNA helicases are critical components of DNA replication, recombination, and repair machinery in all eukaryotes and bacteria. Eukaryotic RecQ helicases are known to associate with numerous genome maintenance proteins that modulate their cellular functions, but there is little information regarding protein complexes involving the prototypical bacterial RecQ proteins. Here we use an affinity purification scheme to identify three heterologous proteins that associate with Escherichia coli RecQ: SSB (single-stranded DNA-binding protein), exonuclease I, and RecJ exonuclease. The RecQ-SSB interaction is direct and is mediated by the RecQ winged helix subdomain and the C terminus of SSB. Interaction with SSB has important functional consequences for RecQ. SSB stimulates RecQ-mediated DNA unwinding, whereas deletion of the C-terminal RecQ-binding site from SSB produces a variant that blocks RecQ DNA binding and unwinding activities, suggesting that RecQ recognizes both the SSB C terminus and DNA in SSB⅐DNA nucleoprotein complexes. These findings, together with the noted interactions between human RecQ proteins and Replication Protein A, identify SSB as a broadly conserved RecQ-binding protein. These results also provide a simple model that explains RecQ integration into genome maintenance processes in E. coli through its association with SSB.

Synergic and Opposing Activities of Thermophilic RecQ-like Helicase and Topoisomerase 3 Proteins in Holliday Junction Processing and Replication Fork Stabilization

“…2, C and D); the main products of the Hel112 reaction on the HJ were Y-shaped forks, whereas only a limited amount of ssDNA was occasionally observed. Several mesophilic RecQ family helicases show the ability to unwind synthetic HJs, but in most cases, their reactions proceed up to production of ssDNA and are stimulated by ssDNA-binding proteins (37)(38)(39)(40). We reasoned that this difference might be due to spontaneous reannealing of the ssDNA products at 55°C.…”

Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance

“…Direct SSB-Ct binding and SSB-stimulated DNA unwinding activity have been described for both RecQ and PriA (8,10,11). However, the fold that binds SSB in ExoI is not conserved in RecQ, nor is it predicted to be conserved in PriA, which leaves open the question of whether the inhibitors can act on these SSB/protein complexes.…”

SSB Antagonizes RecX-RecA Interaction

“…C-terminally deleted variants of SSB, in which the C-terminal domain (up to 42 residues) has been removed, bind somewhat more tightly to ssDNA than does wild type SSB (77,78). In trials to date, no ssDNA binding deficiency (relative to wild type SSB) has been detected with the SSB⌬C8 variant (76,79). Four of the final eight residues of E. coli SSB are aspartate residues, and many proteins interact with this acidic SSB C terminus, (73, 79 -91).…”