The mechanism of Single strand binding protein–RecG binding: Implications for SSB interactome function

Abstract

The Escherichia coli single-strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single-stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide-oligonucleotide binding folds (OB-fold) present in SSB and its interactome partners. Not surprisingly, partner OB-fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB-fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C-terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles.First, and consistent with the proposal of Dixon, to regulate the structure of the C-terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB-folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB-folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB-fold genome guardians identified in prokaryotes. K E Y W O R D SDNA helicase, OB-fold, PXXP motif, RecG, SH3 domain, single-strand binding protein, SSB interactome

SSB facilitates fork substrate discrimination by PriA

“…The mechanism for this is unknown but could involve SSB to SSB transfer. Once there, SSB plays its important roles in effecting the outcome of events at a fork as shown previously for Rep, UvrD and for RecG (40,46,47,52,70). This makes sense because SSB loads PriA onto the DNA and remodels the helicase in the process and there is also a functional interaction between SSB and PriA (20,(35)(36)(37)(38)(39)(40)51).…”

“…This follows because there are two SSB components to the catalytic efficiency enhancement. First, there is direct ssDNA binding to the lagging strand template and second, there is an interaction between the SSB and PriA that involves linker/OB-fold binding (40,45). Binding of SSB to the lagging strand blocks access to this ssDNA thereby ensuring that PriA can only bind to the fork in one orientation.…”

“…As for several other proteins at the replication fork such as RecG, the physical interaction is mediated via the linker domain of SSB and the OB-fold in PriA (40).…”

“…This stimulation involves both a physical and functional interaction between the two proteins (36–39). As for several other proteins at the replication fork such as RecG, the physical interaction is mediated via the linker domain of SSB and the OB-fold in PriA (40).…”

Effects of Conserved Wedge Domain Residues on DNA Binding Activity of Deinococcus radiodurans RecG Helicase

“…In E. coli , SSB-RecG interactions occur via the WD of RecG and the PXXP motifs within the SSB linker domain. In particular, the SSB binding site on RecG overlaps the residues of the binding site for the leading strand arm of the fork in RecG ( Ding et al, 2020 ). Thus, it is likely that these different features do not enable DrSSB to deliver EcRecG to the fork with an efficiency equal to that of DrRecG, which may explain why EcRecG does not confer resistance to Δ recG ( Figure 4B ).…”