The proliferation of all cells relies on the accurate and timely copying of DNA by large macromolecular assemblies termed replisomes. Cellular replisomes are complex, multi-functional assemblies that co-localize a number of essential biochemical functions to increase replication efficiency and speed (O'Donnell et al., 2013). One such function is the unwinding of parental chromosomes to expose template DNA for leading and lagging strand synthesis, a process carried out by specialized, ring-shaped enzymes known as helicases. In Gram-negative bacteria, an enzyme known as DnaB (named DnaC in Gram-positive organisms) serves as the principal helicase for driving replication fork progression (reviewed in Chodavarapu and Kaguni, 2016). DnaB proteins form hexameric rings and are generally incapable of spontaneously associating with chromosomal replication start sites (origins). As a consequence, DnaB helicases are specifically recruited and deposited onto single-stranded origin DNA by dedicated initiation and loading factors.

Replicative helicase loading in bacteria depends on at least one of two known types of proteins. One widespread class, comprising factors related to a protein known as DciA, is ATP-independent (Brézellec et al., 2016; Mann et al., 2017). The second class is composed of DnaC/DnaI-family proteins, which are ATP-dependent and present in model organisms such as E. coli and Bacillus subtilis (B. subtilis) (Brézellec et al., 2016). Both DnaC and DnaI consist of an N-terminal helicase-binding domain (HBD) fused to a C-terminal AAA+ (ATPases Associated with various cellular Activities) nucleotide-binding fold (Figure 1A; Ludlam et al., 2001; Neuwald et al., 1999b). The AAA+ region of DnaC contains numerous signature sequence motifs (Walker A, Walker B, Sensor I and II, Arginine-finger), several of which have been reported to be critical for the function of the loader in E. coli (Makowska-Grzyska and Kaguni, 2010; Mott et al., 2008). A few of these elements have been examined and found to be important for controlling aspects of DnaC activity in vitro as well (Davey et al., 2002; Makowska-Grzyska and Kaguni, 2010; Mott et al., 2008); however, a systematic analysis of their individual mechanistic impact on the efficiency of DNA loading and unwinding by DnaB has been lacking.

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DnaC-type loaders help place DnaB hexamers on single-stranded DNA by opening the helicase ring and allowing a nucleic acid strand to laterally enter into the pore of the complex (Arias-Palomo et al., 2019). In the context of DnaB, six DnaC protomers form a cracked hexameric oligomer, generating composite ATPase active sites at five of the six subunit interfaces (Figure 1B,C; Arias-Palomo et al., 2013; Arias-Palomo et al., 2019). The AAA+ fold of DnaC binds ssDNA directly, at a site distal from its ATP-binding locus (Arias-Palomo and Berger, 2015; Ioannou et al., 2006; Mott et al., 2008). Prior to binding DNA, DnaC protomers latch onto a DnaB hexamer through their HBDs to promote helicase ring opening. In this configuration, electron microscopy studies have shown that DnaB resides in an ADP-like state and DnaC in an ATP-bound form (Arias-Palomo et al., 2013). DNA binding induces a large conformational rearrangement in the DnaB hexamer and compresses the DnaC ATPase ring, switching the ATPase status of the helicase and the loader into ATP and ADP forms, respectively. Curiously, although DnaB helps promote the local co-association of neighboring DnaC AAA+ domains, the ATPase activity of the loader does not manifest until ssDNA is bound (Arias-Palomo et al., 2013; Davey et al., 2002; Kobori and Kornberg, 1982). How DnaB promotes DnaC self-assembly yet does not directly trigger ATPase turnover by the loader is not understood. How DNA binding is relayed to the DnaC ATPase site and whether DnaB ATPase function is important for loading (as structural work has suggested) are similarly unknown. Addressing these questions is important for understanding how replicative helicase loaders exploit nucleotide-coupling mechanisms to generate productive action on client substrates, a question relevant to AAA+ ATPases in general.

Here, we have shown through a structure-guided biochemical approach that defects in different DnaC ATPase elements have distinct impacts on the ability of the loader to deposit DnaB onto DNA and to activate helicase unwinding of forked substrates. DnaC is shown to be capable of promoting helicase unloading in an ATP-dependent manner; both loading and unloading are additionally found to be influenced by the ATPase activity of the helicase as well. We have identified a set of residues in DnaC that couple loader ATPase activity to the binding of DnaB and ssDNA and that unexpectedly modulate the responsiveness of DnaC to a replication restart factor, and we have shown that analogs of one of these elements, an active site arginine, are present in related AAA+ superfamily members that operate with switch-like properties. Our data collectively explain how the ATPase status of DnaC is coupled to the recognition of its DnaB and ssDNA substrates to ensure tight coupling of helicase loading and DNA unwinding with nucleotide turnover.

Cells rely on ATP-dependent protein/protein and protein/nucleic-acid complexes to support a diverse array of critical biochemical processes. A group of enzymes known as AAA+ ATPases are exemplars of such molecular ‘machines,’ participating in events ranging from DNA replication and gene expression to proteolytic homeostasis and vesicle trafficking (Neuwald et al., 1999b). How AAA+ enzymes couple ATP turnover to movement and function is a broadly studied mechanistic question. Although the appropriate engagement of client substrates is clearly important for ATPase coupling, the elements responsible for these interactions are typically far removed (20–30 Å) from the nucleotide-hydrolysis center. The structural linkage elements that relay information over a distance between client occupancy and ATPase status are rarely known.

Here, we have used structure-guided mutagenesis and biochemical analyses to dissect how DnaC, a bacterial AAA+ ATPase that supports the deposition of the replicative DnaB helicase onto DNA, coordinates its ATPase cycle with helicase loading and activation. The opening of the DnaB ring and its subsequent engagement with DNA can occur independently of ATP hydrolysis by DnaC (Figure 2), a finding in accord with previous data showing that neither ATP binding nor the DnaC ATPase domains are required for DnaB loading in vitro (Arias-Palomo et al., 2013; Davey et al., 2002). However, ATP turnover does improve the efficiency of helicase loading (Figure 2), an observation in agreement both with early work of Wahles et al., 1988 (which reported that an ATP-hydrolysis step is important in the binding of the DnaBC complex to DNA) and with a study showing that the replication factor C (RFC) clamp loader becomes compromised for DNA binding when its D or E subunit bears a mutated Arg-finger (Johnson et al., 2006). Interestingly, the ability to hydrolyze nucleotides also has a potentiating effect on the capacity of DnaC to activate DNA unwinding by DnaB (Figure 4). Indeed, hydrolysis-defective DnaC mutants are less capable of activating DnaB than an ATP-binding (Walker-A, K112R) mutant. Moreover, all ATPase mutants are substantially compromised for activating DNA unwinding compared to wild-type DnaC or the helicase-binding domain of DnaC alone. These observations indicate that the HBD is an independent helicase-activating element regulated by its associated ATPase module and that ATP turnover by the loader is important for allowing DnaB to shift into a translocation-competent form.

Recent images of DNA-free and DNA-bound structures of the DnaBC complex have revealed that the nucleotide status of the helicase flips from an ADP state to an ATP configuration when DNA is present (Arias-Palomo et al., 2019). This observation suggested to us that the loading of DnaB onto DNA might be impacted by the helicase’s ATPase function. In testing this idea with a double Walker-A/Walker-B (KAEA) mutant of DnaB, the ability of the helicase to interact productively with nucleotides indeed impacts DNA loading (Figure 3A). Although this coupling is not a strict dependency, a role for nucleotide turnover by DnaB is reminiscent of the need for ATP by the eukaryotic Mcm2-7 replicative helicase to support its loading onto DNA by the origin recognition complex (ORC) (Coster et al., 2014). Interestingly, we found that DnaC can also drive helicase unloading as well as loading (Figure 3D). In contrast to eukaryotes, where unloading of the Cdc45/Mcm2-7/GINS helicase is facilitated by ubiquitylation of MCM7 and removal of the CMG helicase by CDC48/p97 (Deng et al., 2019), relatively little is known about how DnaB helicases are removed from DNA following replication termination in bacteria. Additional studies will be needed to assess a potential role for DnaC in this process.

Structural work has shown that the AAA+ fold of DnaC associates with DNA directly (Arias-Palomo et al., 2019). Three residues—Phe146, Ser177, and Tyr179—form a small DNA-binding locus on the part of the DnaC ATPase domain that faces the interior pore of the dodecameric DnaBC complex; mutation of these residues ablates DnaB loading by DnaC (Arias-Palomo et al., 2019). In examining this region further, we found a fourth amino acid, Lys143, that also participates in binding DNA. Lys143 is generally a lysine or arginine in DnaC/DnaI homologs, and a prior genetic screen Hupert-Kocurek et al., 2007 noted that the integrity of this residue is important for E. coli viability. Here, we have shown that Lys143 is important for supporting the ATPase and DnaB loading activities of E. coli DnaC (Figure 5). The inability of a DnaC^K143E mutant to facilitate DnaB loading onto ssDNA—even though it has an intact HBD and stably binds DnaB—suggests that the alteration allows the loader to maintain a persistent, open-ring state of the helicase. In addition, we found that Lys143 and other amino acids in the DNA-binding patch are critical for DnaC to stimulate fork unwinding by DnaB (Figure 5E, Figure 5—figure supplement 1A–B). Biochemical studies show that disruption of this residue and other amino acids in the region block DNA binding (Figure 5—figure supplement 1C; Arias-Palomo et al., 2019), thereby short-circuiting the ability of the nucleic acid substrate to stimulate ATP hydrolysis by DnaC when the loader is bound to DnaB. Overall, the integrity of the DNA-binding patch on DnaC supports the ability of ssDNA to activate all known DnaC functions, even though it is situated over 20 Å away from the DnaC ATPase center.

A long-standing mechanistic question has been to understand why DnaB is required for stimulating ATP turnover by DnaC, yet is incapable of supporting this activity unless ssDNA is also present (Davey et al., 2002). Unlike a majority of AAA+ ATPases, E. coli DnaC is a monomer on its own (Galletto et al., 2004b) and forms stable oligomeric interactions only when templated by a pre-existing DnaB hexamer (Kobori and Kornberg, 1982). Formation of a higher-order oligomer state is generally a prerequisite for AAA+ ATPase function, as the active site of one subunit typically depends on the contribution of an arginine or lysine from a partner subunit to support nucleotide turnover (Neuwald, 1999a; Neuwald et al., 1999b). Prior work has shown that mutation of the so-termed ‘Arg-finger’ residue to alanine in E. coli DnaC does not interfere with the ability of the loader to deliver DnaB to oriC, but does interfere with DnaC-dependent DNA replication in vitro and DnaC function in vivo (Makowska-Grzyska and Kaguni, 2010). Here we have shown that this amino acid is critical for DnaC ATPase activity (Figure 2A) and that while it is only moderately important for DnaB loading, its presence is necessary to activate DNA unwinding by the helicase (Figures 2D and 4C).

Interestingly, DnaC possesses a second arginine (Arg216) just upstream of its Arg-finger (Arg220). This amino acid is essentially invariant among DnaC/DnaI homologs (Figure 6A). Prior studies have shown that the integrity of this residue is unnecessary for DnaC to bind ATP or associate with DnaB but is important for supporting replication in vitro and for cell viability (Makowska-Grzyska and Kaguni, 2010). Here, we have shown that Arg216 is actually an essential element for coupling substrate DNA binding to loader ATPase activity. Mutation of the amino acid to aspartate severely impairs DnaC’s ATPase activity and its ability to mobilize and activate DnaB on DNA. However, substitution with alanine not only increases the rate of ATP hydrolysis by DnaC, but also allows nucleotide turnover to occur even in the absence of DNA (Figure 6B). The DnaC^R216A mutant also overrides the ATPase defect imparted by a DNA-binding mutant (e.g., K143E), yet does not degrade the ability of DnaC to assist with either DnaB loading or the stimulation of DnaB-mediated fork unwinding. Inspection of a DNA-free DnaBC structure reveals that Arg216 is positioned where the nucleophilic water would be expected to reside prior to ATP hydrolysis; in the presence of ssDNA, this amino acid shifts away to allow access to the reactive γ-PO₄ (Figure 8A–B). Thus, Arg216 acts not just as a substrate/ATPase coupler, but also as a steric governor, preventing the DnaC ATPase from firing when oligomerized by DnaB to prevent futile cycling until substrate DNA is bound. The action of the Arg-coupler explains how the ATPase activity of DnaC allosterically responds to the binding of ssDNA at a region distal to the active site. The binding of ssDNA to the tetrad residues elicits a subtle rocking and compression motion between neighboring DnaC protomers (Arias-Palomo et al., 2019); this action leads the Arg-coupler to shift out of the active site, releasing its repressive action on ATPase function (Figure 1—video 1). In this regard, the Arg-coupler acts in a manner akin to the ‘glutamate switch’, a residue found in many AAA+ ATPases that controls the conformation of the catalytic glutamate in response to ligand binding (Zhang and Wigley, 2008). Interestingly, a polar amino acid at the position of the glutamate switch is present in E. coli DnaC (Thr134), but it does not make ideal hydrogen-bonding interactions with the catalytic glutamate (Glu170) in the repressed (DNA-free) ATPase state (Figure 8—figure supplement 1A). Instead, Thr134 forms a contact with an acidic amino acid (Asp 189), which is in turn hydrogen-bonded to the Arg-finger. Neither the glutamate switch threonine nor its acidic anchor residue is particularly well conserved among DnaC/DnaI homologs, although these two amino-acid positions are typically occupied by polar residues (Figure 8—figure supplement 1B–C). Whether the glutamate switch has been repurposed to control the Arg-finger instead of the catalytic glutamate in bacterial helicase loaders remains to be determined.

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In examining the activity of the Arg-coupler mutant, we also probed the activity of a DnaC mutant (E176G) encoded by the dnaC810 allele. Purified DnaC810 has been shown to load DnaB on SSB-coated ssDNA and on D-loops, bypassing the need for the PriA replication restart protein, and it has been proposed that the DnaB loading activity of this mutant is mediated by a cryptic DNA-binding site in DnaC (Xu and Marians, 2000). Inspection of the structure shows that Glu176 sits on a loop that connects one-half of the DNA-binding tetrad (Ser177 and Tyr179) to the Walker-B loop of the ATPase center (Figure 1C). Interestingly, DnaC810 shows a similar pattern of behavior as the alanine substitution of Arg216: DnaB-dependent but ssDNA-independent ATPase activity, and near wild-type efficiency in loading DnaB onto DNA and activating the helicase for fork unwinding (Figure 7). However, unlike the R216A mutant, the dnaC810 allele supports cell viability (Makowska-Grzyska and Kaguni, 2010; Sandler et al., 1996). A comparison of structures of the DnaBC complex in DNA-free and -bound states shows that the Walker-B loop on which Glu176 sits switches between two conformations (Arias-Palomo et al., 2019). Although a structure of DnaC810 bound to DnaB is currently unavailable, it seems likely that the substitution of the glutamic acid with glycine alters the flexibility of the Walker-B region, perhaps mimicking a DNA-bound conformational state to relieve DnaC of the repressive action of Arg216. The finding that the DnaC810 can act in a manner similar to that of the arginine-coupler mutant suggests that tight ATPase control by the loader is necessary to properly coordinate its activity with pathways that resuscitate stalled replication forks. Understanding precisely how this action might allow DnaC⁸¹⁰ and the R216A mutant to act independently of replication restart factors awaits further study.

Given the widespread nature of AAA+ proteins, we were curious as to whether the coupling mechanisms we observed for DnaC might be found in other ATPase family members. Because AAA+ ATPases recognize diverse types of substrates—single- and double-stranded nucleic acids, protein chains—it seemed unlikely that there would be any consensus to their substrate-recognition sites. However, DnaA, a DnaC paralog that serves to reorganize and melt origins to initiate replication in bacteria, does retain a highly conserved arginine in the same position as the Arg-coupler. Sequence analyses show that two proteins used to catalyze replicative helicase loading in eukaryotes, Orc4 and Orc1, also retain such an element (Figure 8C). Interestingly, these proteins all belong to the same ‘initiator’ subgroup, or clade, of AAA+ proteins, suggesting that the coupling arginine seen in DnaC may play a similar role in regulating substrate-dependent ATP turnover in these proteins as well. In the case of DnaA, the integrity of the equivalent of DnaC-Arg216 (Arg281 in E. coli DnaA) has been shown to be dispensable for ATP binding and melting of oriC DNA, but necessary for forming a sufficiently stable complex that can support DnaB loading by DnaC (Felczak and Kaguni, 2004); by comparison, mutation of the neighboring Arg-finger of DnaA, R285A, leads to a failure in unwinding oriC (Kawakami et al., 2005). These findings echo the retention of certain specific activities but an overall functional defect seen for DnaC^R216A. Interestingly, although there is no known data on the phenotypic effect of mutating the equivalent residue in human Orc4 (Arg205), mutation of the analogous residue in Orc1 (Arg666, to Trp) has been linked to Meier-Gorlin syndrome (Guernsey et al., 2011). It has yet to be determined biochemically whether Arg-coupler mutations in ORC might also lead to an unlinking of ATPase activity from DNA-binding status (as is seen for DnaC) or to a different functional defect.

Other proteins in the initiator clade, such as IstB (a transposition helper protein and another DnaC paralog), do not possess a clear homolog of the Arg-coupler, nor do several archetypal AAA+ ATPases of other clades (e.g., clamp loader, classic, pre-Sensor I insert, etc). A review of several representative structures of these proteins nonetheless does reveal a potential common theme shared between DnaC, IstB, and clamp loaders. In addition to blocking access to the reactive γ-PO₄ of bound nucleotide, the Arg-coupler of DnaB-bound DnaC also engages the polar Sensor I amino acid (Figure 8D) and releases this interaction when DNA is bound. The Sensor I amino acid is a conserved feature of AAA+ proteins and is generally important for ATP hydrolysis (Hattendorf and Lindquist, 2002; Neuwald et al., 1999b; Steel et al., 2000). In the case of IstB— whose ATPase is primed by DNA binding but not actuated until it engages its client IstA transposase—a DNA-bound structure of the protein shows that the Arg-coupler is replaced with a small amino acid (e.g., alanine), but that a bulky amino acid (tyrosine) on the Walker-B loop occupies the same general position to occlude the γ-PO₄ moiety and remodel the Sensor I loop (Figure 8E; Arias-Palomo and Berger, 2015). Clamp loaders, whose AAA+ clade is closely related to that of the initiators, also lack the upstream Arg-coupler; however, in the presence of a client clamp—which is necessary but insufficient for stimulating ATPase activity on its own—access to the γ-PO₄ is again occluded by a Sensor I interaction with a positively charged residue on an adjacent helix (Figure 8F). A comparison of clamp loader structures reveals that an arginine or lysine in this highly conserved central helix can, upon DNA binding, swing out to release the Sensor I residue, thereby allowing the Arg-finger to access the γ-PO₄ (Bowman et al., 2004; Gaubitz et al., 2020; Simonetta et al., 2009). Interestingly, an N-terminal motif known as ‘RFC box II’ also has been shown in the archaeal clamp loader system to be important for linking ATP hydrolysis to clamp loading: when ablated, the loader shows elevated ATPase activity but is compromised for clamp loading, a behavior similar to that of the DnaC Arg-coupler (Seybert et al., 2006). An interesting distinction of initiators and clamp/helicase/transposase loaders compared to other AAA+ systems is that they are switch-like in their behavior, as opposed to acting in a more processive manner. Restricting access to γ-PO₄ of ATP and sequestering the Sensor I amino acid may thus serve as a general fail-safe in these systems, as it does in DnaC, to allow an active ATPase center to be assembled while avoiding premature hydrolytic events. Whether other switch-like AAA+ proteins have evolved similar regulatory mechanisms remains to be determined.

All data generated during this study is included in the manuscript and supplementary files.

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Author details

1. Neha Puri

   Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, United States

   Present address

   FogPharma, Cambridge, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Amy J Fernandez

   Competing interests

   is affiliated with FogPharma. The author has no financial interests to declare.

   "This ORCID iD identifies the author of this article:" 0000-0003-0079-7489

2. Amy J Fernandez

   Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Neha Puri

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6130-3326

3. Valerie L O'Shea Murray

   1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, United States
   2. Saul Ewing Arnstein & Lehr, LLP, Centre Square West, Philadelphia, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review and editing

   Competing interests

   is affiliated with Saul Ewing Arnstein & Lehr, LLP. The author has no financial interests to declare.

4. Sarah McMillan

   Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, United States

   Contribution

   Formal analysis, Investigation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

5. James L Keck

   Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, United States

   Contribution

   Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

6. James M Berger

   Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   jberge29@jhmi.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0666-1240

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