Proteins whose function is to maintain the integrity of the genome and safeguard it are classified as genome guardians. Many guardians such as the DNA helicase RecBCD and the recombinases RecA and Rad51 contain neither OB-folds nor SH3 domains (Chen et al., 2008; Conway et al., 2004; Singleton et al., 2004). However, the number of genome guardians utilizing OB-folds to mediate changes in DNA is increasing (Figure 3) (Agrawal and Kishan, 2003; Bochkarev and Bochkareva, 2004; Flynn and Zou, 2010; Gao et al., 2018; Nguyen et al. 2020). Recent work has shown that the SSB interactome is the first family of OB-fold genome guardians identified in E. coli (Bianco, 2021). However, SSB interactome members are not the only OB-fold proteins guarding the bacterial genome as shown for RuvA, which is not an interactome partner but contains OB-folds (Rafferty et al., 1996). In eukaryotes, the OB-fold family of genome guardians is large and likely to increase in size as additional structures are determined (Flynn and Zou, 2010). Thus the concept of OB-fold genome guardians is universal and at present includes at least 40 proteins and this list is likely to grow.

Representative members of the prokaryotic and eukaryotic OB-fold genome guardian families are shown in Figure 3. Included in this figure are the canonical single-strand DNA binding proteins, SSB in E. coli, and RPA and the human SSB1 complex from eukaryotic cells. There are also nucleases (Exo I and RecJ), recombination mediators (BRCA2 and RecO), DNA ligases, polymerases (Pol II), and helicases (PriA, RecG, and the MCM complex) as well as telomere end-binding (CTC1—part of the CST complex, POT1, TPP1, and TAP82) and branch migration complex proteins (RuvA). Visible inspection of the proteins selected, reveals that the number of OB-folds per polypeptide varies from one to as many as seven and the substrate-binding partner capabilities of each domain present per protein complex is also variable. This is perhaps best exemplified by CTC1 which has a total of 7 OB-folds (Lim et al., 2020). The first 4 have no demonstrated substrate specificity; the fifth or OB-fold E, binds protein exclusively and OB-folds 6 and 7 bind to telomere ssDNA and, ssDNA and protein, respectively. There are also examples of OB-folds such as those in SSB and RPA that bind to DNA non-specifically including tails of G4 quadruplexes, whereas POT1 and CTC1 proteins bind to sequence-specific ssDNA in telomere ends with high affinity (Wold and Kelly, 1988; Kim et al., 1992; Nandakumar et al., 2010; Ray et al., 2013; Rice et al., 2017; Shastrula et al., 2018; Lim et al., 2020). For the CST complex (which contains CST1) its DNA substrate-specificity is length-dependent: specific when ssDNA is short and non-specific as DNA length increases (Miyake et al., 2009; Hom and Wuttke, 2017). SSB, RPA70, POT1, and CTC1 contain OB-folds that bind to ssDNA but SSB and RPA bind to both ssDNA non-specifically and proteins, and, like SH3 domains, the SSB OB-folds also bind to acidic phospholipids (Fan and Pavletich, 2012; Zhao et al., 2019; Bianco, 2021). Within the domains that bind proteins, the mechanism of binding also differs, with some binding in the cleft formed between the RT and nSrc loops (POT1 and SSB) and others being partially wrapped by the binding partner (BRCA2) (Yang et al., 2002; Bianco et al., 2017; Chen et al., 2017; Rice et al., 2017; Ding et al., 2020). Finally, there are examples of proteins that bind to duplex DNA and again, their mechanism of binding is distinct. For RuvA, it binds to Holliday junctions while DNA ligases bind to nicked duplexes and MCM proteins bind to both ss- and dsDNA. This is explained in more detail in the next section (Iwasaki et al., 1992; Parsons et al., 1992; Ellenberger and Tomkinson, 2008; Tomkinson and Sallmyr, 2013; Shi et al., 2018).

The variability in OB-fold types is utilized by genome guardians to orchestrate the myriad of protein-DNA and protein-protein interactions required to maintain the integrity of the genome (Flynn and Zou, 2010; Amir et al., 2020). In the sections that follow, the mechanism of substrate binding by OB-folds and the ways that genome guardians use this binding to protect the genome are discussed. As there are so many genome guardians, it is not possible to discuss all possibilities. Instead, key proteins for which structures and biochemistry are available have been selected to highlight how the core of the OB-fold is used to guard the integrity of the genome (Arcus, 2002; Agrawal and Kishan, 2003; Theobald et al., 2003; Richard et al., 2009; Ashton et al., 2013; Wu et al., 2016; Nguyen et al., 2020; Bianco, 2021).

The variation in OB-folds suggests that proteins containing these domains may bind to DNA substrates in distinct ways to effect different reaction outcomes while maintaining genome integrity. To demonstrate how this can occur, 4 genome guardians were selected. The first is the E. coli SSB protein which is the canonical single-strand DNA binding protein (Meyer and Laine, 1990; Lohman and Ferrari, 1994). The second is DNA ligase III which binds to a nicked duplex and facilitates the sealing of the nick (Cotner-Gohara et al., 2010; Simsek and Jasin, 2011). RuvA, like SSB, is a tetramer but instead of having the OB-folds exposed to accommodate ssDNA, the folds are centrally located and interact with dsDNA during branch migration (Ariyoshi et al., 2000; Yamada et al., 2002). Fourth, the MCM DNA helicase forms a ring-shaped structure, and like RuvA, the OB-folds are positioned in the center of the ring and contact the ssDNA (Froelich et al., 2014).

E.coli SSB is the most well-studied single-strand DNA binding protein (Chase and Williams, 1986; Meyer and Laine, 1990; Kowalczykowski et al., 1994; Lohman and Ferrari, 1994; Shereda et al., 2008; Bianco, 2021). The role of this protein is to bind to exposed ssDNA and to as many as twenty partners that constitute the SSB interactome to regulate their activities concerning genome stability (Costes et al., 2010; Ryzhikov et al., 2011; Yu et al., 2016; Huang and Huang, 2018). The active form of SSB is a stable homo-tetramer (Sancar et al., 1981). Each monomer is divided into two domains defined by proteolytic cleavage: an N-terminal domain comprising the first 115 residues and a C-terminal tail spanning residues 116 to 177 (Curth et al., 1996). The tail is comprised of an intrinsically disordered linker and acidic tip (Lohman and Ferrari, 1994; Shereda et al., 2008; Kozlov et al., 2015; Bianco, 2017). For further details see the section “OB-fold regulation” including Figure 6.

The N-terminal domains are visible in all crystal structures to date, are responsible for tetramer formation, and are almost exclusively OB-fold [Figures 2A, 4A; (Raghunathan et al., 2000; Savvides et al., 2004)]. ssDNA binding by this domain is non-specific and occurs via the wrapping of the polynucleotide around the SSB tetramer using an extensive network of electrostatic and base-stacking interactions with the phosphodiester backbone and nucleotide bases, respectively [Figure 4A and (Chrysogelos and Griffith, 1982; Kuznetsov et al., 2006; Raghunathan et al., 2000)]. Within this complex, ssDNA bound to the tetramer is wrapped and bound securely in the OB-folds where it is protected (Figures 4A,B). In addition to ssDNA binding, OB-folds are also responsible for binding to the linker region of nearby SSB tetramers resulting in cooperative ssDNA binding (Bianco, 2017; Ding et al., 2020). The linker, which has not been visualized in crystal structures to date, mediates protein-protein interactions using a mechanism similar to that employed by SH3 domains binding to PXXP ligands (Kaneko et al., 2008; Su et al., 2014; Huang and Huang, 2018; Nigam et al., 2018; Ding et al., 2020). Linker binding by the SSB OB-fold, its competition with ssDNA for binding, and the role this plays in protein function will be elaborated in the section “OB-fold regulation”.

DNA ligase III functions in nuclear and mitochondrial DNA replication and repair pathways (Sallmyr et al., 2020). Like other ATP-dependent eukaryotic DNA ligases and the widely used T4 enzyme, DNA ligase III contains a common catalytic region consisting of a nucleotidyltransferase domain and an OB-fold [Figure 4C; (Ellenberger and Tomkinson, 2008; Shi et al., 2018; Tomkinson and Sallmyr, 2013) (Cotner-Gohara et al., 2010)]. In addition, the enzyme also possesses an α-helical DNA binding domain that is critical to the DNA clamping mechanism (see below). In sharp contrast to SSB, these three domains encircle nicked, double-strand DNA with each making contacts with the duplex, thereby sequestering the 3′-OH and 5′-PO₄ (Ellenberger and Tomkinson, 2008; Shi et al., 2018). This clamping or jackknife mechanism is conserved in other ligases and holds the dsDNA in a distorted conformation where the DNA is bent, underwound and the minor grove adjacent to the nick is significantly widened [Figure 4D; (Cotner-Gohara et al., 2010)]. The OB-fold via its RT loop binds to the minor groove opposite the nick, secures the DNA within the active site of the nucleotidyltransferase domain, and functions to position the nicked DNA substrate during all the remaining steps of the ligation reaction (Pascal et al., 2004; Cotner-Gohara et al., 2010). Thus for DNA ligase III, the role of the OB-fold is to participate in the jackknife mechanism and to bind to the minor groove of the duplex thereby positioning the DNA so that efficient ligation can occur.

The RuvA tetramer is intrinsic to the branch migration process catalyzed by RuvAB (West, 1997). In contrast to both SSB and DNA ligase III, it binds to intact DNA in the form of a Holliday junction (HJ). RuvA has several roles which include (i) changing the configuration of a Holliday junction to an open-square structure that is energetically more favorable for branch migration; (ii) targeting RuvB to the junction and stimulating its DNA helicase activity; (iii) coupling strand separation to duplex rewinding and (iv), facilitating binding of RuvC leading to resolution. Structural analysis of RuvA reveals that the protein consists of three domains. Domains I and II constitute the core of the protein and are responsible for tetramer formation and HJ binding [Figure 4F; (Ariyoshi et al., 2000; Nishino et al., 1998)]. Domain III is flexible, is not visible in the structures shown, interacts with RuvB, and modulates its ATPase and consequently its branch migration activity (Nishino et al., 1998; Nishino et al., 2000). Each RuvA monomer contains a single, N-terminal OB-fold in Domain I, with each contributing an acidic pin, comprised of residues E55 and D56, crucial to the branch migration process [Figures 4F–I; (Ingleston et al., 2000)].

HJs are dynamic structures that fluctuate between at least four different conformations in the presence of divalent metal cations, one of which is shown in Figure 4 between panels F and G (Hyeon et al., 2012; Joo et al., 2004; McKinney et al., 2003; Wyatt and West, 2014). RuvA binding halts these conformational dynamics converting the HJ into an open planar configuration a requirement for efficient branch migration [Figures 4G,I; (Gibbs and Dhakal, 2018; Lushnikov et al., 2003; Panyutin et al., 1995)]. In this configuration, the extended nSrc loop of each RuvA monomer is positioned in the center of the HJ in preparation for strand separation coupled to rewinding during the branch migration process (Figure 4H). Concurrently, the HJ is inclined 10° upwards from the ideal plane on the surface of RuvA (Figure 4I). Once two RuvB hexamers are bound to opposite ends of the RuvA tetramer, branch migration ensues and requires a screw motion and lateral pulling or pumping of the dsDNA, which passes through the center of the RuvB hexamers, and over the surface of the tetramer. Here RuvA uses the 4 acidic pins comprised of E55 and D56 contributed from the n-Src loop of each OB-fold to direct the path of each nascent single DNA strand through the complex (Stasiak et al., 1994; Rafferty et al., 1996; Ariyoshi et al., 2000; Ingleston et al., 2000; Putnam et al., 2001). Thus in this context, the RuvA OB-folds are providing an additional catalytic function to the RuvAB complex, that is strand separation and rewinding coupled to ATP hydrolysis-coupled dsDNA translocation by the RuvB hexamers.

The MCM proteins form a hexameric ring that in archaea is comprised of six identical subunits while in eukaryotes, the complex is a heterohexamer with subunits arranged in a specific order (Davey et al., 2003; Maiorano et al., 2006; Tye, 1999). The MCM complex assembles with five other subunits comprised of Cdc45 and GINS (Go, Ichi, Nii, andSan; five, one, two, and three in Japanese; consisting of Sld5, Psf1 (partner of Sldfive 1), Psf2 and Psf3), to form the replicative DNA helicase, Cdc45-MCM-GINS or CMG (Ilves et al., 2010). For all MCMs the OB-folds of each subunit are positioned within the center of the channel where they can interact with both ds- and nascent ssDNA (Figure 4J). Two parts of the OB-fold facilitate these interactions. The extended RT-loop of the OB-fold of MCM subunits is interrupted a 3₁₀-helix which is itself interrupted by a Zn-finger (Figure 2C). A recent structure of the budding yeast S. cerevisiae CMG bound to a forked DNA revealed that the zinc fingers of each MCM, extend from the complex to contact the unwound duplex DNA ahead of the MCM ring (Yuan et al., 2020). The nascent unwound ssDNA interacts with the canonical OB-fold where highly conserved arginine residues extend from the barrel of the OB-fold and are thereby positioned in the center of the channel to make contact with the nascent ssDNA (Figure 4K). Thus in this case the OB-fold contacts both ss and dsDNA.

Modifications to the central β-barrel structure of the OB-fold allow proteins to bind to and modify DNA in a variety of ways that were unlikely to have been predicted when the structures of the first OB-folds were determined (Murzin, 1993). If left unregulated, DNA binding by these proteins could have disastrous consequences for genome stability as they could cause excessive strand separation and/or spurious melting of duplex DNA that otherwise might be lethal to the cell as suggested previously (Pant et al., 2004; Shokri et al., 2006; von Hippel and Delagoutte, 2001). It follows then, that binding must be regulated. This can be achieved in different ways with three examples of protein/OB-fold binding presented.

The shelterin complex is responsible for maintaining the integrity of telomeres (de Lange, 2005). In humans, this complex consists of six subunits, TRF1, TRF2, TIN2, RAP1, POT1, and TPP1 (Diotti and Loayza, 2011) Of these, POT1 and TPP1 contain OB-folds that are relevant to this section (Figure 3) (Liu et al., 2004; Theobald and Wuttke, 2004; Wang et al., 2007). POT1 and TPP1 function together by forming a stable heterodimer that protects chromosome ends and regulates telomerase activity (Wang et al., 2007; Xin et al., 2007). These two proteins bind one another via the protein binding domain of TPP1, also known as the POT1-binding motif (Figure 5A, left) (Chen et al., 2017; Rice et al., 2017). This interaction is crucial to POT1 function as it enables its localization to the telomere as well as regulating its binding. The structure of this complex reveals that, in addition to other interactions, the C-terminal one-third of the POT1 binding motif of TPP1 binds to the third OB-fold of POT1 (Figure 5A). A 3₁₀-helix is located in the canonical, OB-fold ssDNA binding groove positioned between the RT and nSrc loops. The binding of TPP1 to POT1 stabilizes POT1 (Chen et al., 2017). This interaction is disrupted by mutations, with one of these, Q623, located within the POT1 OB-fold binding site for TPP1 (Figure 5A, right panel). When POT1-TPP1 binding is eliminated, POT1 becomes unstable with a shorter half-life, resulting in lower protein levels coupled to an activated DNA damage response at telomeres (Chen et al., 2017).

In mammalian cells, BRCA2 is a large and intricate example of OB-fold regulation within a single, multi-functional protein (et al. 2002; Thorslund and West, 2007; Thorslund et al., 2010; Shahid et al., 2014). BRCA2 binds to multiple protein partners and to DNA, to mediate the repair of DNA double-strand breaks and inter-strand cross-links by RAD51-mediated homologous recombination (Dray et al., 2006; Saeki et al., 2006; Le et al., 2021).

The structure of the C-terminal domain of the protein which is critical for the interaction with DNA revealed how binding and regulation could occur (Yang et al., 2002). This region of BRCA2 protein contains a helical domain and 3 OB-folds, with one interrupted by what has been called the tower domain (Figure 5B). The tower consists of two long, antiparallel helices capped by a three-helix bundle that has been proposed to bind dsDNA within the context of a tailed duplex (Schneider et al., 1998; Yang et al., 2002; Thorslund et al., 2010). The three OB-folds lie in close linear proximity, with two of them bound to ssDNA (red) and the third bound to the Deleted in split-hand/split-foot syndrome protein (DSS1; black). DSS1 is an intrinsically disordered, 70-residue peptide involved in multiple cellular functions including DNA repair (Marston et al., 1999; Kojic et al., 2003; Kragelund et al., 2016; Schenstrom et al., 2018). It is required for BRCA2 stability and the control of BRCA2 function in homologous recombinational repair (Li et al., 2006; Zhou et al., 2009). In the absence of DSS1, recombinational repair is virtually eliminated and this is due to increased degradation of BRCA2 (Li et al., 2006; Kristensen et al., 2010).

The binding of BRCA2 to an ssDNA/dsDNA junction is mediated by OB-folds 2 and 3 and likely the tower domain. The OB-folds bind to ssDNA while the tower is proposed to bind duplex DNA. This binding facilitates the nucleation of RAD51 filaments on the single-stranded tails of a processed, dsDNA break that are bound by RPA (Thorslund et al., 2010; Zhao et al., 2015). In addition to stabilizing BRCA2, DSS1 functions as an allosteric effector of BRCA2 and not as a DNA mimic as proposed (Alagar and Bahadur, 2020; Le et al., 2020; Zhao et al., 2015; Zhou et al., 2009). Here DSS1 binding to OB-fold 1 and the adjacent helical region results in structural changes in the C-terminal domain as well as the conversion of BRCA2 dimers into monomers (Alagar and Bahadur 2020; Le et al., 2020). It is conceivable that these effects are linked, but this has not been demonstrated. Using molecular dynamics simulations, Algar and Bahadur showed that the binding of DSS1 to the C-terminal tail of BRCA2 stabilizes this region (Figures 5B,C) (Alagar and Bahadur, 2020). This follows because apo BRCA2 (not bound to either DSS1 or DNA) showed a greater level of fluctuations in the helical domains and OB-folds 1 and 2, relative to the DSS1-BRCA2 complex. The effect of binding of DSS1 to OB-fold 1 may be propagated to OB-fold 2 and the tower, resulting in the restriction in conformational changes. In summary, the binding of an intrinsically disordered peptide to one OB-fold results in stabilization of protein structure and this influences both BRCA2 activity and possibly DNA binding as well.

The second example of an intrinsically disordered protein regulating OB-fold function is seen in the prokaryotic SSB interactome (Bianco, 2021; Lecointe et al., 2007; Shereda et al., 2008). Here, a 20-member, OB-fold, DNA-binding protein family is regulated by one member, the SSB protein whose OB-folds are in turn, controlled by acidic phospholipid, ssDNA, and protein binding in a competitive fashion (Ding et al., 2020; Harami et al., 2020; Zhao et al., 2019). The key region of SSB regulating interactome function is the intrinsically disordered linker or linker, which is positioned between the OB-fold and acidic tip of the protein (Figure 6, Key and inset top right). Here, the linker uses one or more of its conserved PXXP motifs to mediate protein-protein interactions by binding to the canonical ssDNA binding pocket positioned between the RT and nSrc loops of the OB-folds in either SSB or interactome partners where it competes with ssDNA (Figure 6, insets bottom right and bottom left). This binding forms the essence of the linker/OB-fold model while the tip functions as a regulator of the tail region and as a secondary protein binding site (Bianco, 2021; Ding et al., 2020; Zhao et al., 2019). The binding mechanism employed in the linker/OB-fold model to regulate the SSB interactome is similar to that used by SH3 domains to bind PXXP motifs to mediate target protein function (Bianco et al., 2017; Saksela and Permi, 2012; Yu et al., 1994). This follows because SH3 domains are structurally almost identical to OB-folds and, there are 3 PXXP motifs in the linker region of each SSB monomer (Figures 1C, 6, inset top right) (Agrawal and Kishan, 2001; Bianco, 2017).

The binding of SSB to ssDNA results in a conformational change in the protein so that the C-termini are more exposed (Kozlov et al., 2010; Williams et al., 1983). When ssDNA binding involves multiple tetramers, it occurs cooperatively and results in shortening of the DNA length (Chrysogelos and Griffith, 1982; Krauss et al., 1981; Ruyechan and Wetmur, 1975). The change in ssDNA length occurs because the polynucleotide is wrapped around each tetramer (Figure 4A). Concurrently, each tetramer also binds to its neighbors via linker/OB-fold interactions (Figure 6, center). Within this complex, some OB-folds bind to DNA while others bind exposed linker PXXP-motifs of adjacent tetramers [Figure 6, lower right; (Bianco, 2017; Ding et al., 2020)]. This results in an extensive network of linker/OB-fold interactions forming a stable complex that protects the ssDNA requiring elevated concentrations of salt or translocation by DNA motor proteins to disrupt them (Figure 6, center) (Lohman and Ferrari, 1994; Manosas et al., 2013; Green et al., 2016; Bianco, 2017; Bianco et al., 2017). Not surprisingly, mutation of the PXXP motifs eliminates cooperative binding to ssDNA (Ding et al., 2020).

The conformational change in the protein associated with binding of SSB to ssDNA also makes linkers available for interactome partner binding which facilitates these proteins being loaded onto the DNA, their functions regulated, and, in some cases, this is accompanied by SSB dissociation (Bell et al., 2015; Sun et al., 2015; Bianco et al., 2017; Nigam et al., 2018; Ding et al., 2020; Hwang et al., 2020; Wang et al., 2020). One example of an interactome partner is the RecG DNA helicase which binds to, and regresses stalled DNA replication forks into Holliday junctions (McGlynn et al., 2001; Singleton et al., 2001; Manosas et al., 2013; Lloyd and Rudolph, 2016; Bianco, 2020).

RecG has a single OB-fold in the wedge domain, responsible for fork binding (Mahdi et al., 1997; Singleton et al., 2001). This OB-fold binds to the linker of SSB, resulting in loading of the helicase onto DNA concomitant with the remodeling of RecG (Bianco et al., 2017; Ding et al., 2020; Sun et al., 2018; Sun et al., 2015; Tan et al., 2017). When the key residues of the linker/OB-fold interface, namely the PXXP motifs of SSB or separately, the OB-fold of RecG are mutated, SSB-RecG binding is eliminated (Ding et al., 2020). It is worth noting that when those residues that are part of the binding site of the helicase for the leading strand arm of the fork are mutated, SSB binding is reduced as much as 25-fold and fork binding is also eliminated (Figure 6, inset lower left) (Bianco and Lyubchenko, 2017; Briggs et al., 2005; Singleton et al., 2001). This is consistent with the model that that these binding sites overlap and that DNA and SSB binding is competitive (Bianco and Lyubchenko, 2017; Briggs et al., 2005; Sun et al., 2015). As the PriA and RecO OB-folds are essential for SSB binding, the linker/OB-fold model likely applies to all SSB interactome members which have an OB-fold as proposed (Figure 3; left side) (Kozlov et al., 2010; Inoue et al., 2011; Ryzhikov et al., 2011; Bianco et al., 2017; Ding et al., 2020; Hwang et al., 2020; Bianco, 2021).

The RecG and PriA DNA helicases are members of the SSB interactome, the first family of OB-fold genome guardians identified in prokaryotes (Bianco, 2021). Each of these proteins binds to SSB via linker/helicase OB-fold interactions, resulting in the loading of these enzymes onto stalled (RecG) or regressed (PriA) DNA replication forks, concomitant with their remodeling (Buss et al., 2008; Sun et al., 2015; Yu et al., 2016; Ding et al., 2020; Wang et al., 2020). These DNA helicases also use their OB-folds to bind to and alter or remodel the fork structure in unique ways.

RecG catalyzes fork regression, where a stalled DNA replication fork is moved in a backward direction, away from the site of DNA damage, resulting in the formation of a Holliday Junction (Figure 7A). To do this, the helicase domains bind to the parental duplex DNA ahead of the fork while the OB-fold binds to the fork with high affinity (Figure 7B) (Singleton et al., 2001). The helicase domains use the energy from the hydrolysis of ATP and product release to generate >35pN of force to push the OB-fold through the DNA (Manosas et al., 2013). The OB-fold then couples DNA strand separation to duplex rewinding both in the wake of the advancing enzyme as well as ahead of it, resulting in Holliday junction formation (Figure 7C). This process occurs at an average rate of 269 ± 2bp/sec and processivity of 480 ± 20 bp (Manosas, Perumal, Bianco, Ritort, Benkovic and Croquette 2013). How the RecG-OBfold binds to forks is distinct from that of the MCM hexamer. For RecG, the barrel interacts with and splits the arms of the fork to facilitate strand separation followed by rewinding [Figure 7B; (Singleton et al., 2001)]. For MCMs, the extended RT-loop binds to duplex DNA while the OB-fold barrel binds to nascent ssDNA [Figures 2C, 4J,K; (Meagher et al., 2019)].

PriA binds to forks once RecG has catalyzed regression and/or additional processing has taken place to restore the fork structure (Marians 1999; Marians 2000). In contrast to RecG, PriA uses its OB-fold to bind to the 3′-OH group on the nascent leading strand arm of the restored fork with high affinity (Figure 7D, red base) (Mizukoshi et al., 2003; Sasaki et al., 2007) (Mizukoshi et al., 2003; Nurse et al., 1999). This binding is critical to both the activation of the ATPase activity as well as efficient ATP hydrolysis and is significantly enhanced by SSB (Manhart and McHenry, 2013; Tan and Bianco, 2021; Tanaka et al., 2007). This serves to enhance the ability of PriA to discriminate the correct fork structure by as much as 140-fold, orienting the DNA helicase on the fork so that it can unwind the nascent lagging strand arm (Figure 7E). Duplex unwinding ensures that the preprimosome (a complex of PriA, PriB, DnaT, PriC, DnaB, and DnaC) can be loaded onto the template lagging strand and that replication restart proceeds in the correct direction (Lee and Marians, 1987; Jones and Nakai, 1999; Jones and Nakai, 2001). This involves the loading by PriA, of the replicative DNA helicase, DnaB onto the lagging-strand template via a complex series of protein-protein interactions reminiscent of primosome (preprimosome + primase) assembly for ϕX174 DNA (Masai and Arai, 1996; Jones and Nakai, 1999; Marians, 1999; Marians, 2000).