Structural snapshots of the mechanism of ATP-dependent DNA damage recognition by UvrA

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: accept

Abstract
Nucleotide excision repair is a DNA repair pathway which detects and fixes various DNA lesions that distort the structure of DNA. In bacteria, the pathway starts with the UvrA protein which has two adenosine triphosphate modules and forms dimers. The DNA is handed over from UvrA to UvrB, which is a weak helicase that verifies the presence of damage. Despite intense studies, the role of the ATPase activity of UvrA in damage recognition is unclear. Here, we present a series of cryo-electron microscopy structures of UvrA in complex with three different DNAs and in the presence and absence of nucleotides. We also present a structure of UvrA:UvrB:DNA complex. These structures reveal a major rearrangement of the UvrA dimer upon ATP binding. We propose that these conformational changes are used to mechanically probe the integrity of DNA for damage localization. Collectively, our results present snapshots of UvrA’s ATP-dependent DNA damage detection.

Introduction
Nucleotide excision repair (NER) is one of the most versatile DNA repair pathways involved in fixing diverse structurally unrelated DNA lesions1–5. Bacterial NER involves three key proteins: UvrA, UvrB, and UvrC1,6. UvrA detects the DNA damage and hands it over to UvrB for verification. UvrA then dissociates, and UvrC nuclease is recruited, which executes cuts on both sides of the lesion. The excised fragment is removed by UvrD helicase, and the resulting gap is filled by DNA polymerase I1.

UvrA is a dimeric protein that belongs to the adenosine triphosphate (ATP)-binding cassette (ABC) family of ATPases, which also contains ABC transporters7, the mismatch repair protein MutS8,9, and the DNA repair proteins RecF10 and Rad5011. Crystal structures of B. stearothermophilus UvrA, M. tuberculosis UvrA, and D. radiodurans UvrA2 revealed structural organization of the protein12–14. UvrA contains two ATP-binding domains with insertion of the signature domains (Fig. 1a). The N-terminal signature domain contains two additional insertions: one is responsible for interactions with UvrB, and the other is involved in DNA binding. There are two composite ATPase active sites in each UvrA monomer. The proximal active site is formed by the N-terminal ATP-binding domain and C-terminal signature domain, and the distal by the C-terminal ATP-binding domain and N-terminal signature domain13,14 (Fig. 1a).

A crystal structure of T. maritima UvrA in complex with modified DNA revealed the mechanism of the preferential binding of UvrA to modified DNA15, with only the deformed conformation of the DNA complementary to the UvrA surface. The DNA in the complex is bent, unwound, and stretched in the region around the modification. Such deformations are often observed in damaged DNA (e.g., in the case of a cis-syn thymine dimer16). UvrA’s damage recognition has been proposed to rely on a combination of sensing helix flexibility and pre-existing deformations that are caused by the presence of the lesion15.

A key element of the mechanism of action of UvrA is its ATPase activity. Bacterial strains in which either ATPase active site is defective in ATP binding because of replacement of the key lysine residue in the Walker A motif to alanine are extremely UV-sensitive17, implying ATPase activity is required for the recognition of UV-induced lesions. However, its exact role is unknown.

After UvrA detects a lesion, the DNA is passed to the UvrB protein for damage verification. UvrB belongs to helicase superfamily 218–20. Its ATPase activity is only activated in the presence of UvrA and DNA21. UvrB inserts its characteristic β-hairpin between the two DNA strands, clamping one of them22. We previously used modeling and negative-stain electron microscopy (EM) reconstruction to propose a model of the bacterial UvrA:UvrB:DNA complex and mechanism of damage verification23. We postulated that the UvrA dimer recruits two UvrB molecules, and each UvrB clamps a different DNA strand under the β-hairpin and translocates towards the lesion. Only undamaged DNA can be accommodated under the hairpin, so the UvrB molecule that clamps the damaged strand will stall in front of the lesion while the other will dissociate. The stalled UvrB then recruits the UvrC protein to execute precise cuts on the damaged strand. This mechanism explains how the lesion’s presence is verified and how UvrA’s initial approximate, symmetrical, and non-strand-specific damage localization converts into a precise incision of the damaged strand23.

In the present study, we characterize the conformational changes of UvrA during the ATPase cycle through a series of cryo-electron microscopy (cryo-EM) structures of UvrA:DNA complexes, in different nucleotide-bound and nucleotide-free states. The structures reveal major rearrangements of the UvrA dimer that are induced by ATP binding and imply a role of ATPase activity in damage recognition through the mechanical probing of DNA deformations. We also report a cryo-EM structure of the UvrA:UvrB:DNA complex.

Results
UvrA:DNA structures without nucleotide or with ADP
We sought to understand how UvrA’s ATPase activity (i.e., nucleotide binding, hydrolysis, and release) is involved in DNA damage recognition. To this end, we wanted to determine cryo-EM structures of various complexes of UvrA with DNA and nucleotides (Table 1, Supplementary Table 1, 2 and Supplementary Figs. 2–11, 13). We used UvrA proteins from several bacterial species and obtained the best results with the enzyme from Clostridium thermocellum (Ct) (Supplementary Fig. 1a). We verified that the Ct-UvrA and Ct-UvrB are able to promote incisions of modified DNA by UvrC (Supplementary Fig. 1b).

For structural studies, we used three DNAs. The first was based on a well-studied substrate for bacterial NER (a 50 bp duplex with a single fluorescein-modified thymine [flu-dT] paired with cytosine in the middle of its sequence)21,24,25. flu-dT is a mimic of DNA damage that is efficiently processed by UvrABC machinery in vitro and has been extensively used in biochemical studies22,25,26. The second DNA contained a single abasic site in the middle of the sequence. This modification should have a destabilizing effect on the DNA structure and should increase its deformability. UvrA binds avidly to DNA containing an abasic site as compared to non-damaged DNA, so we used this modification as another model DNA recognized by UvrA. It needs to be noted that UvrB is poorly loaded on abasic site DNA and thus this lesion is not incised efficiently27. However, bacterial NER can repair base-excision repair-resistant chemically heterogeneous abasic sites28–32. Finally, we also employed a regular unmodified 50 bp DNA. All these DNAs were mixed with Ct-UvrA, initially in the absence of nucleotide. The complexes were purified by the pull-down of His-tagged UvrA and analyzed by cryo-EM.

The first cryo-EM reconstruction we obtained was for a complex of Ct-UvrA with flu-dT-DNA and without nucleotide. We determined initial reconstructions using samples prepared with and without glutaraldehyde cross-linking. The resulting maps from the non-crosslinked samples were very similar to those from the cross-linked samples. However, the resolution of the maps from the cross-linked samples was higher (3.1 Å resolution, Supplementary Fig. 2). So, for the preparation of other complexes the cross-linking step was included. In the Ct-UvrA:flu-dT-DNA reconstruction, we could observe a well-defined density for the ATP binding-I and -II domains, and the signature-I and -II domains. Lower-resolution density was observed for the DNA-binding domains, and no density was visible for UvrB-binding domains (Fig. 1b, c). We also observed a lower resolution density for DNA, showing two fragments of double-helical regions that are located between and interact with ATP binding-II and signature-II domains of each subunit of the UvrA dimer. The DNA is also bound by the two DNA-binding domains (Fig. 1b, c) with contacts mostly mediated by van der Waals interactions and involving residues S309, N315, T390, V394 and M395. In addition, side chain of R386 is missing in the model, but based on its vicinity to the DNA, we predict it would also bind the nucleic acid.

The two halves of the DNA density are coaxial (Fig. 1c, d), indicating that the DNA in the Ct-UvrA:flu-dT-DNA complex is not bent. The poor quality of the density in the middle of the DNA prevented us from determining whether this DNA is stretched and unwound, and the exact extent of these deformations. However, when we superimposed a computer-generated model of ideal B-form DNA on one half of the DNA in our structure, it did not overlap with the other portion of the DNA, which was not used in the superposition (Fig. 1d). This implies that the middle portion of the DNA in our structure is deformed, most likely unwound. This may result from a preexisting deformation in flu-dT DNA or the induced fit of the damaged DNA with higher deformability to the surface of UvrA.

Synthetic DNA duplexes can interact with UvrA in an arrangement in which only the terminal part of the DNA is bound by the protein and the blunt end located in the middle of the UvrA dimer33. However, this reconstruction is unlikely to represent the “end-binding”, because we observed continuous residual density in the middle of DNA in an unsharpened map (Supplementary Fig. 14a). We cannot also fully exclude the possibility that two blunt DNA ends form stacking interactions, which would result in the formation of a pseudohelix with continuous DNA density. However, it has been shown that in the absence of nucleotide, ~80% of UvrA molecules bind on damage33, so, likely, this is the binding mode we observe as well.

We also obtained a 3.8 Å cryo-EM reconstruction of Ct-UvrA in complex with abasic DNA in the absence of any nucleotide (Supplementary Fig. 3, 15a). For this complex, to exclude particles that may correspond to “end-binding”, we used a subset of particles, identified by 3D classification, producing a clear continuous density in the center of the DNA in the unsharpened map (Supplementary Fig. 14b). The obtained reconstruction was essentially identical to the reconstruction with flu-dT-DNA, showing that in the absence of the nucleotide, the complex adopts the same conformation regardless of the type of helix-destabilizing modifications that are present (Supplementary Fig. 15a, c).

The next structure we determined was for Ct-UvrA in complex with unmodified DNA in the absence of nucleotide, determined at 4.4 Å resolution. This reconstruction showed that in the absence of nucleotide, the UvrA:DNA complex adopts the same conformation as in the complexes with flu-dT and abasic DNAs. However, for this reconstruction, we did not observe a clear density in the center of the DNA in the unsharpened map. The 3D classification that focused on the center of DNA did not yield any subset of particles with a clear density between the two halves of the double helix. Therefore, we could not exclude the possibility of “end-binding” for the complex with unmodified DNA, so this structure was not further analyzed.

For ABC ATPases, the largest conformational changes occur upon ATP binding and hydrolysis34, so we expected that the ADP-bound form of UvrA would be very similar to the nucleotide-free form. To verify this, we determined a structure of the Ct-UvrA:flu-dT-DNA complex in the presence of ADP at 3.9 Å resolution (Supplementary Fig. 4, 15b). In the reconstruction, we observed a clear density for the ADP at all four active sites of UvrA (Supplementary Fig. 16a). As expected, the resulting structure of the UvrA:DNA complex adopts essentially the same conformation as in the nucleotide-free state (Supplementary Fig. 15b, d). However, the density of the DNA was better defined in the presence of ADP than in the absence of nucleotide (Supplementary Fig. 14c). 3D Flex analysis35 in cryoSPARC showed that in the ADP complex, the mobility of the DNA was smaller than in the no-nucleotide sample (Supplementary Movie 1). This lower mobility could explain the better resolution of the DNA map.

All the structures described above have very similar conformations, so for further analyses, we selected one of them, the Ct-UvrA:flu-dT-DNA complex structure, which was solved at the highest resolution (Table 1). We compared it with previously determined crystal structures of Bs-UvrA in complex with ADP (PDB ID: 2R6F)13, Mt-UvrA (PDB ID: 3ZQJ)12, and Tm-UvrA in complex with a self-annealing 32 bp DNA that contained two flu-dT residues (Tm-UvrA:flu-dT(2)-DNA, PDB ID: 3PIH)15; (Fig. 1e and Supplementary Fig. 15e–g). In the Tm-UvrA:flu-dT(2)-DNA structure, the DNA is bent, stretched, and unwound, and pyrophosphate molecules were observed at active sites in positions that corresponded to β and γ phosphates of bound ATP. The superpositions showed that the overall conformation of the UvrA dimer was similar for all of the analyzed crystal structures and our cryo-EM structure of the Ct-UvrA:flu-dT-DNA complex (Fig. 1e and Supplementary Fig. 15e–g). For example, when the Tm-UvrA dimer structure was superimposed on the Ct-UvrA dimer, the resulting root mean square deviation (RMSD) was 4.4 Å over 1,472 C-α atoms and 1.5 Å over 678 C-α atoms, if only ATP-binding-I and -II domains were used for superposition. Despite the similarity of the UvrA dimer conformation, we observed pronounced differences in the conformation of the DNA between the cryo-EM structure of Ct-UvrA:flu-dT-DNA and the crystal structure of Tm-UvrA:flu-dT(2)-DNA (see Discussion). Briefly, the trajectory of the DNA in the Ct-UvrA structure is straight, while it is bent in the Tm-UvrA structure (Fig. 1c, e). Another clear difference between the Ct- and Tm-UvrA structures is the positions of the DNA-binding domains. In the Tm-UvrA structure, they are located further away from the DNA and do not interact with it (Fig. 1e). This implies that these domains interact with the DNA only when it adopts a straight conformation.

In summary, in the absence of nucleotide or presence of ADP, the UvrA dimer that interacts with the DNA adopts a conformation that is very similar to the previously determined crystal structures of both the DNA-free and DNA-bound forms. We term this the “basal” conformation.

Structures of UvrA in complex with DNA and AMPPNP
We next sought to understand the effect of ATP binding on the conformation of UvrA and its interactions with DNA. For the ABC family of ATPases, ATP binding induces the largest changes in protein conformation34. To capture the ATP-bound state, we used AMPPNP (a non-hydrolyzable analog of ATP). We obtained a 3.1 Å resolution reconstruction of the Ct-UvrA:flu-dT-DNA:AMPPNP complex (Supplementary Fig. 5) and built its atomic model. In the reconstruction, we observed a density for ATP binding-I, ATP binding-II, signature-I, signature-II, and linker domains and a clearly defined density for the double helix of the bound DNA (Fig. 2a, b). This density was best defined in the region that interacted with UvrA (Supplementary Fig. 14f). We also observed clear densities for AMPPNP molecules at all four ATPase active sites (Supplementary Fig. 16b). No density was observed for the UvrB-binding domain, and a poor density was observed for the DNA-binding domain, implying their mobility. Therefore, we did not build these regions in the atomic model.

Comparisons of the Ct-UvrA:flu-dT-DNA structures in the absence of nucleotide and in the presence of AMPPNP revealed profound rearrangement of the dimer structure (Fig. 2c, d and Supplementary Movie 2). AMPPNP binding to ATPase active sites induces a movement of signature domains toward the ATP-binding domains in both ATPase modules. This has several consequences. The UvrA dimer interface formed by ATP-binding domain I is rearranged. Signature I domains and ATP-binding domains II are rotated as one structural unit around axes that are roughly perpendicular to the axis of the DNA, with the former domain undergoing the largest change. This change creates clefts between the signature I domains and ATP-binding domains I from two different subunits of the dimer. These clefts now accommodate the characteristic β-hairpin elements from the dimer subunits that contribute the ATP-binding domain I to the cleft formation. This is possible due to the rotation of the signature domain II toward the center of the UvrA dimer, as the β-hairpin element is a part of the signature domain II. Because the most extensive interactions with the DNA are mediated by the signature domain II, its movement is accompanied by the movement of the two halves of the DNA duplex and its bending (Supplementary Movie 2). We named the conformation of UvrA that was observed in the presence of AMPPNP “ATP-bound conformation 1”.

During the conformational change, the dimerization interface between the two ATP binding-I domains is altered (Fig. 2c, d). In the absence of nucleotide, interactions between the ATP binding-I domains from the two subunits of the dimer are between the following residue pairs: R54-L507, S58-I85, Y55-Y55, Y55-A84, Y55-P83, Y55-I85, F66-F66, G80-M508, L81-L507, P83-L507 and P83-V510. These interactions are rearranged in the presence of AMPPNP and involve the following pairs: Y55-Y55, Y55-S82, Y55-P83, S58-P83, A63-L67 and F66-F66 (Fig. 2d). In the AMPPNP-bound structure the side-chains of Y55 and F66 are missing in the model, but we predict their interactions based on the vicinity with other residues. The buried solvent accessible surface that was calculated using UCSF ChimeraX for the ATP binding-I domain within the dimer is reduced from ~900 Å2 in the absence of AMPPNP to ~500 Å2 in the presence of the nucleotide.

The terminal portions of the DNA duplex remain bound between the ATP-binding-II domain and signature-II domain of each promoter, and, remarkably, the same protein-DNA contacts are observed as in the nucleotide-free structure (Supplementary Fig. 17e). The result of the change in arrangement of the UvrA dimer, particularly the inward rotation of the signature-II domain, is that the two DNA halves that are observed in our structure are no longer coaxial, and the duplex is bent by ~20° (Supplementary Movie 2). We predict that conformational changes in the DNA also involve stretching and unwinding in the middle. The exact magnitude of these changes cannot be precisely quantified because the density in the middle portion of the DNA was less well-defined in our reconstructions (Supplementary Fig. 14f). For the same reason, we cannot exclude the possibility that during the conformational change in the UvrA dimer, the DNA register changes (i.e., the DNA slides at the protein-nucleic acid interface). In the Ct-UvrA:flu-dT-DNA:AMPPNP reconstruction, we did not observe a clearly defined density of the DNA-binding domains, which confirms that when the DNA adopts a bent conformation, it no longer interacts with these domains, and they become mobile.

We next determined a structure of UvrA with unmodified DNA in the presence of AMPPNP (Supplementary Fig. 7). Analysis of the cryo-EM data led to the identification of three complex conformations, with approximately uniform distribution of particles between them. One subset of particles corresponded to the basal conformation. The density of the DNA was poorly defined and visible only in the vicinity of the ATP binding-II and signature-II domains, with no clear density in the center of the DNA. Therefore, in this reconstruction, we cannot exclude “end binding” and it was not further analyzed. The second conformation corresponded to ATP-bound conformation 1, similar to the one that was determined for the complex with flu-dT DNA and AMPPNP (Fig. 3b, e). Finally, a new conformation was observed. We term this “ATP-bound conformation 2” (Fig. 3c, f). The main difference between ATP-bound conformations 1 and 2 is that the interface between the two monomers of UvrA in the latter is more similar to the basal conformation (Fig. 3c, f). The DNA is bent by ~21°. We carefully inspected the maps in the center of the DNA for both the ATP-bound conformations and observed a continuous density of the DNA backbone (Supplementary Fig. 14h, j). So, these reconstructions are unlikely to represent the “end-binding”. The ATP-bound conformation 1 can be converted to conformation 2 by the rotation of UvrA dimer subunits relative to each other (Supplementary Movie 2). During this rotation, the DNA contacts are maintained, so it is likely accompanied by overwinding of the DNA duplex.

In the ATP-bound conformation 2 reconstruction, we observed density near the ribose ring of nt 17 present at the center of the DNA. This density is located between the backbone of S61 and the side chain of F755 and could correspond to a flipped-out base. However, adding the base to our atomic model resulted in steric clashes with the protein. Therefore, we decided to insert an abasic nucleotide in the structure in this position. Further structural studies and the determination of higher resolution structures will be required to visualize the base flipping in this position.

We also determined structures of the Ct-UvrA:abasic-DNA:AMPPNP complex at 2.7 and 3.0 Å resolution (Supplementary Fig. 6). Each corresponded to approximately 40% of particles. One was nearly the same as ATP-bound confirmation 1 that was determined for the complex with flu-dT DNA and AMPPNP (Supplementary Fig. 17a, c), and the other corresponded to ATP-bound conformation 2 (Supplementary Fig. 17b, d).

One interesting consideration is why ATP-bound conformation 1 dominated in our cryo-EM sample for flu-dT DNA, whereas two ATP-bound conformations were observed for abasic and unmodified DNA. This may be attributable to different deformability or preexisting deformations in each of the DNAs we used. flu-dT can be the most flexible DNA in our set; when UvrA binds it, it readily adopts ATP-bound conformation 1. For abasic DNA, its different dynamic properties can drive the formation of both ATP-bound conformations 1 and 2. As described above, cryo-EM potential maps of ATP-bound conformation 2 suggest binding of a flipped out base by UvrA. Perhaps this conformation is preferred on DNAs in which the modification allows such binding of a flipped out base. In sum, the ability of UvrA to adopt different ATP-bound conformations may facilitate the recognition of different DNA lesions.

In both ATP-bound conformations (1 and 2), the bending of the DNA brings its middle portion closer to the UvrA dimer interface. This, together with the rearrangement of this interface, results in new protein-DNA interactions that have not been observed previously. In particular, side chains of Q65 and aromatic side chains of Y62 and F66 from the ATP binding-I domain are inserted in the center of the double helix and form van der Waals and stacking interactions with nucleobases (Fig. 4a). Intriguingly, this element in the ATP-bound conformation 2 is inserted even deeper into the DNA, forming additional contacts that are mediated by N270 and N271 (Fig. 4b). We term this protein element a “wedge”. Its insertion leads to flipping out of two central bases. It also destabilizes the DNA, explaining why the double helix is not well-defined in the center in our reconstruction. The likely purpose of this insertion is to actively probe the stability of the DNA, particularly the strength of base pairing, which would be reduced for a damaged duplex. However, it is possible that this complex does not represent the entire engagement of the DNA damage.

To verify whether the wedge-DNA interaction results in flipping out of the bases at the center of the DNA, we used DNA that comprised a 2-Aminopurine base in the middle of the sequence. 2-Aminopurine increases its fluorescence when it is removed from the helical stack (e.g., by flipping out). We used two DNAs that corresponded to the 50 bp DNAs that were used for structural studies. In the first, the 2-Aminopurine was placed opposite of the flu-dT residue (AP-1). In the second, 2-Aminopurine oligonucleotide was hybridized with unmodified DNA (AP-2). We also used control dsDNAs, in which 2-Aminopurine was placed close to one of the termini in the region that does not undergo flipping (AP-3) (Fig. 4c). We measured 2-Aminopurine fluorescence at 370 nm for dsDNA alone and upon the addition of UvrA in the absence/presence of AMPPNP or ADP. The results showed that 2-AP fluorescence for AP-1 and AP-2 DNAs increased by a small amount upon the addition of UvrA without nucleotide or in the presence of ADP (Fig. 4d, e). In the presence of AMPPNP, fluorescence increased approximately two-fold for AP-1 (Fig. 4d) and five-fold for AP-2 (Fig. 4e). The increase was much higher for AP-2 (DNA without flu-dT residue). AP-1 corresponds more closely to flu-dT oligo in our structural studies and AP-2 to unmodified DNA. In our structures, UvrA:flu-dT-DNA complex predominantly adopts ATP-bound conformation 1, while for the complex with unmodified DNA, conformations 1 and 2 are observed. In each ATP-bound conformation, the environment of the flipped-out base is different. In ATP-bound conformation 2 it may be stabilized by tight protein interactions (the unmodeled density described above). This different environment could explain different change of the signal for the flipped out 2-AP for the two DNAs. No change in fluorescence was observed for AP-3 (Fig. 4f). These results show that centrally located bases are flipped out upon DNA binding by UvrA in the presence of AMPPNP.

We also tested UvrA variants in which the wedge residues were replaced with alanine: Y62A, F66A, and Y62A/F66A. They either did not show any change in fluorescence or showed a smaller change than that observed for the wild type protein, confirming that these residues play a role in flipping out bases at the center of the DNA (Fig. 4d, e). Collectively, these results confirmed that UvrA uses the wedge element to probe the structural integrity of the DNA upon its bending associated with ATP binding.

We also determined structures of Ct-UvrA:flu-dT-DNA:AMPPNP complex using two variants of UvrA, which are unable to bind ATP at either active site36 (K39A and K647A) and structures of wild-type UvrA without DNA in the presence and absence of AMPPNP (Supplementary Figs. 8–11). In all these structures, the UvrA dimer adopted basal conformation (Supplementary Fig. 18). This means that DNA binding and the presence of AMPPNP at both active sites promote the formation of ATP-bound conformations by UvrA.

In summary, we determined structures of UvrA in complex with two types of modified DNA and with an unmodified duplex in the presence and absence of AMPPNP. These structures showed a large rearrangement of the UvrA dimer to two new ATP-bound conformations, accompanied by conformational changes in the DNA.

Configuration of ATPase active sites and their coordination
DNA binding affect the ATPase activity of UvrA37. One study found that upon the addition of undamaged DNA, UvrA’s ATPase activity was reduced by ~50%, and with damaged DNA, it was at the same levels as without DNA38. Detailed kinetic studies of nucleotide binding and hydrolysis have shown that in the absence of DNA, the proximal site binds ATP with low affinity and triggers fast ATP hydrolysis in the high-affinity distal site36. In the presence of damaged or undamaged DNA, all four sites perform ATP hydrolysis. Unmodified DNA stimulates both active sites, and adenosine diphosphate (ADP) release from the distal site is accelerated. Damaged DNA slows ATP hydrolysis by the proximal site; at the same time, the distal site performs ATP hydrolysis cycles. These effects lead to the formation of more stable complexes on damaged DNA and more dynamic complexes on native DNA. The structural basis of the coupling of the two ATPase sites and how ATPase activity and DNA binding affect each other are unknown. Having a large set of structural data available, we analyzed the configuration of ATPase active sites in UvrA to elucidate the mechanism of their coordination. The main change at the ABC ATPase active site is when catalytic serine from the ABC signature motif and a nucleotide-binding lysine from the Walker A motif are brought closer together to form a catalysis-conducive configuration. For example, in the Ct-UvrA structures that are described herein, the distance between C-α atoms of the two residues changes from ~12-13 Å to ~10 Å. We term these two states open and closed configurations, respectively. Both catalytic serine and a nucleotide-binding lysine are located at the N-termini of α-helices, so their movement toward each other also requires changes in the position of the two helices. For our analyses, we chose active sites of the Ct-UvrA:flu-dT-DNA:AMPPNP complex as a reference. In this structure, all four active sites are occupied by AMPPNP and present in active (closed) configurations (Fig. 5a, Supplementary Fig. 16b and Supplementary Table 2). For Ct-UvrA in complex with unmodified DNA and abasic DNA in the presence of AMPPNP in both ATP-bound conformations (1 and 2), all active sites are also in a closed configuration (Fig. 5b). As expected, for all DNAs that were used, in the absence of nucleotide or presence of ADP, both active sites of the UvrA monomer are in an open configuration (Fig. 5c, d). Interestingly, for the ADP complex structure, nucleotide density is observed at both proximal and distal active sites that are in the open configuration (Fig. 5d and Supplementary Fig. 16a). This means that ADP binding to either active site does not require a closed configuration. Additionally, for UvrA in the absence of DNA and with or without AMPPNP, both active sites are in the open configuration (Fig. 5e, f). Interestingly, however, in this open configuration, the distal site binds AMPPNP (Fig. 5e). This is in agreement with previous observations that in the absence of DNA, the distal site binds ATP while proximal site binds the nucleotide with very low affinity36. Moreover, pre-steady-state kinetics studies showed that in the absence of DNA in the burst phase of ATPase activity, only two ATPase sites of the dimer perform fast ATP hydrolysis, and the distal sites are responsible for the burst. Adenosine triphosphate binding to the proximal site is required for this activity. This mean that in the absence of DNA, UvrA can transiently undergo a conformational change, perhaps without complete rearrangement of the UvrA dimer, that closes the distal active site and allows low ATPase activity.

We next analyzed the configuration of active sites in the structures of K39A and K647A UvrA variants in the presence of flu-dT and AMPPNP. In both of these structures UvrA dimer adopted a basal conformation. For the K39A variant with a mutation in the proximal active site, both active sites are in an open configuration, and the nucleotide is present in the distal site, similar to the Ct-UvrA:AMPPNP structure (Fig. 5g and Supplementary Fig. 16g). For the K647A variant with a mutation of the distal site, both active sites are empty and in an open configuration (Fig. 5h). These observations indicate that AMPPNP binding to the distal site is independent of occupancy of the proximal site. Conversely, AMPPNP binding to the distal site is required for the proximal site to bind the nucleotide.

In the K39A variant, in which the proximal site is unable to bind ATP, the distal site contains AMPPNP but does not have the catalytic configuration. The ABC signature motif from the signature-I domain is too far from the ATP binding-II domain for the catalytic serine (S490) to engage in catalysis. Upon dimer rearrangement, the distal ATP binding site is organized. The ABC signature motif from the signature-I domain moves closer to the ATP binding-II domain, thereby sandwiching the nucleotide in a conformation that is conducive to ATP hydrolysis. Therefore, dimer rearrangement that requires ATP binding to both active sites enhances activity at the distal active site. A full understanding of the coupling between ATPase active sites will require further studies, including careful molecular dynamics simulations to understand dynamic aspects of this process.

The mechanism of the coupling between DNA binding and ATPase activity
One important element of ABC ATPase active sites is the so-called Q loop with a conserved glutamine residue that interacts with the γ phosphate of the ATP molecule. When basal and ATP-bound conformations of the UvrA dimer that were determined herein were compared, the Q loop was found to adopt a very different conformation (Fig. 6a–c). In the basal conformation, the two conserved glutamines (Q89 and Q690) are far away from the ATPase active sites. In the ATP-bound conformations, they move closer to γ phosphates. Q89 binds AMPPNP at the proximal site and Q690 the nucleotide at the distal site (Fig. 6b, c). Interestingly, the loops that comprise these residues are in the vicinity of the DNA backbone. In the presence of a bent duplex the DNA likely stabilizes the conformation of the loops and positioning of the glutamines, which in turn helps bind ATP at both active sites. This mechanism likely contributes to the enhancement of ATPase activity upon DNA binding36,39,40.

Coupling between DNA binding, ATP binding, ATP hydrolysis, and conformational changes in the UvrA dimer involves another element, namely the positioning of signature-II domains. We previously proposed a mechanism of ATPase activation that relies on movements of these domains because in the Tm-UvrA:flu(2)-dT complex structure, a movement of the signature domain that is induced by DNA binding closes the distal active site. Signature-II domains comprise an insertion with a Zn-finger and a long β-hairpin. The conformation of this element differs significantly in the published structures and those presented herein. The position of this element is mostly determined by the presence and conformation of the DNA. In the absence of the DNA in some of the crystal structures, these Zn-finger/β-hairpin elements are rotated inward toward the center of the UvrA dimer, and their tips interact through stacking interactions of aromatic side chains of phenylalanines (F755 In Ct-UvrA). In ATP-bound conformations with bent DNA presented herein, clefts are formed between the signature I domains and ATP-binding domains I from the other subunit of the dimer. These clefts are formed due to the rotation of signature I and ATP-binding II domains and the rearrangement of the dimer interface. The clefts accommodate the Zn-finger/β-hairpins whose movement is associated with the simultaneous inward rotation of signature II domains. This inward movement of Zn-finger/β-hairpin is likely aided by the interaction of signature-II domains with the DNA, bringing them closer as compared to their configuration observed in the basal conformation. These observations suggest a model in which, when the DNA is bent, Zn-finger/β-hairpin movement promotes formation of the closed configuration of the distal active site and its activation. This, with the additional contribution of stabilization of the position of Q loops, promotes ATP hydrolysis that allows the UvrA dimer to return to its basal conformation, producing cycles of conformational probing of the DNA’s flexibility.

Structure of the UvrA:UvrB:DNA complex
We next sought to determine how UvrA functions in the context of the UvrA:UvrB complex. To this end, we prepared a complex of UvrA and UvrB proteins from C. thermocellum in the presence of 50 bp DNA that harbored a single flu-dT paired with cytosine in the middle of the sequence and AMPPNP. We obtained a 3.0 Å resolution consensus reconstruction of the Ct-UvrA:UvrB:flu-dT-DNA:AMPPNP complex (Supplementary Figs. 12a–e, 19a). In this reconstruction, we observed a density for the following domains of UvrA: ATP binding-I, ATP binding-II, signature-I, signature-II, linker, and UvrB-binding. We also observed densities for two UvrB molecules that flank the UvrA dimer and correspond to domains 1a, 1b, and 2 of UvrB. We then calculated a 3.2 Å focused map for UvrB chains and additionally observed domain 3, however, its density was relatively poorly defined (Supplementary Fig. 19b). We also observed a density for a fragment of the DNA that is bound by the β-hairpin element of UvrB (the density for two nucleotides on top of the β-hairpin element of UvrB was not visible). Finally, we calculated a composite map by combining two focused UvrB maps with our consensus map (Fig. 7a).

Previously obtained structural information about UvrA:UvrB interactions includes a structure of a complex of interacting domains from UvrA and UvrB (PDB ID: 3FPN)41 and UvrA:UvrB complex without DNA (PDB ID: 3UWX)42, both solved for proteins from Geobacillus stearothermophilus. In our previous work23, we combined these structures with the Tm-UvrA:flu-dT(2)-DNA complex (PDB ID: 3PIH)15 and B. caldotenax UvrB (Bc-UvrB) in complex with DNA (PDB ID: 2FDC)22 to model the architecture of UvrA:UvrB:DNA assembly23. This model was verified by low-resolution negative-stain EM. The previously published structures showed two UvrA:UvrB interfaces. The first involves contacts between the UvrB-binding domain of one protomer from the UvrA dimer and domain 2 from UvrB. The second interface is formed between the signature-II domain of the second protomer of the UvrA dimer and domain 1b of UvrB. These interfaces are essentially the same in the present cryo-EM reconstruction of Ct-UvrA:UvrB:flu-dT-DNA:AMPPNP. However, their position relative to each other is changed. This is possible because of flexibility in the positioning of the UvrB-binding domain of UvrA, which is linked to the rest of the structure through a flexible region that comprises a Zn-finger.

The main difference in the architecture of UvrA:UvrB complexes is the arrangement of the UvrA dimer. In our theoretical model, we used the Tm-UvrA:flu-dT(2)-DNA structure, in which the UvrA dimer adopts a basal conformation23. In our current cryo-EM structure, the UvrA dimer adopts ATP-bound conformation 1, which is nearly identical to the Ct-UvrA:flu-dT:AMPPNP structure (Supplementary Fig. 19c). This implies that the UvrA:UvrB complex can be formed for different UvrA dimer conformations. Notably, in the structure of the Gs-UvrA:UvrB complex without DNA, the arrangement of the UvrA dimer is different from all the configurations described herein42. This conformation is incompatible with DNA binding but further shows the flexibility of UvrA:UvrB assembly (Supplementary Fig. 19d).

We next sought to understand the role of conformational changes in UvrA that are induced by nucleotide binding and hydrolysis in the context of the UvrA:UvrB complex. We studied UvrB loading on the DNA, using His-tagged UvrB that was mixed with UvrA and flu-dT or unmodified DNA in the absence of nucleotide and in the presence of ADP, ATP or AMPPNP. We used wild-type UvrB and its K44A variant with a substitution in activating its ATPase activity. We reasoned that blocking UvrB translocation on the DNA could increase the efficiency of the pull-down. Indeed, the pull-down efficiency was slightly better for the K44A variant, so we used it in the subsequent experiments. After mixing UvrA with DNA, UvrB K44A was added. After an incubation, UvrB was immobilized using Ni-beads, which were then washed using a buffer containing 1 M NaCl. The UvrB:DNA complex, in which the β-hairpin clamps one of the DNA strands, has been shown to withstand a high salt concentration43. Therefore, we reasoned that only DNA molecules for which one of the strands is clamped by UvrB will co-purify with UvrB after high ionic strength washes. UvrB was eluted using high imidazole, and fractions were analyzed on SDS-PAGE for protein and TBE-urea PAGE for DNA (Fig. 7c, d). The result showed that the DNA, both flu-dT and unmodified, co-purified with UvrB in the presence of AMPPNP or ATP but not with ADP or in the absence of nucleotide. With ATP or AMPPNP present, small amounts of UvrA also co-purified with UvrB, perhaps stabilized on the DNA by strong binding of the nucleic acid by flanking UvrB molecules. This implies that only ATP-bound conformations of the UvrA dimer and the corresponding bent DNA conformation allow the efficient loading of UvrB on the DNA.

Notably, in the presence of AMPPNP, UvrB loading on the DNA was equally efficient for modified and unmodified DNA (Fig. 7c, d). This observation is in agreement with the previous findings that showed that non-hydrolyzable analogs of ATP attenuate the specificity of UvrA for damaged DNA33,44–46. In the experiment performed in the presence of ATP, we observed much less efficient DNA co-purification of DNA (Fig. 7c, d). We surmise that with ATP hydrolysis cycles occurring, the interaction between UvrA and DNA is much more dynamic, which leads to less efficient DNA pull-down. Importantly, however, flu-dT DNA was pulled down more efficiently than unmodified counterpart showing specificity of the system for modified DNA in the presence of ATP (Fig. 7g, h).

We also performed the pull-down experiments using a variant of UvrA with a substitution in a residue forming the wedge element: Y62A. In the presence of ATP this variant showed impaired UvrB loading on the DNA (Fig. 7e–h). In the presence of AMPPNP both wild-type and Y62A proteins promoted UvrB loading equally efficiently. These results show that the wedge element plays an important role in ATP-dependent DNA damage recognition by UvrA.

Collectively, our results show that conformational changes in UvrA and accompanying deformation of the DNA can also occur in the context of the UvrA:UvrB complex. These rearrangements promote UvrB loading on the DNA such that it clamps one of the DNA strands using its β-hairpin. This suggests a model of DNA damage recognition in which UvrA uses ATP binding and hydrolysis cycles to probe the deformability of the DNA. The loading of UvrB can proceed only if the double helix can be bent. Another possibility is that this can occur in an ATP-independent fashion, where the UvrA dimer binds pre-deformed bent DNA.

Methods
Bacterial strains
In this study, we used the following E. coli strains: BL21 Star (DE3) (Agilent; E. coli F− ompT hsdS [r −m –] gal dcm rne131[DE3]) and Top10 (Thermo Fisher; E. coli F− mcrA Δ[mrr-hsdRMS-mcrBC] φ80lacZΔM15ΔlacX74recA1araD139Δ[ara-leu]7697galUgalKrpsL [StrR]endA1nupG).

Protein preparation
C. thermocellum (Ct; recently reclassified to Acetivibrio thermocellus) UvrA, UvrB and UvrC expression plasmids were prepared based on the pET28 expression vector (Novagen). The Ct-UvrA and Ct-UvrB proteins were overexpressed both with an N-terminal 6×His tag and as a fusion with an N-terminal 6×His tag and SUMO protein in E. coli BL21 (DE3) STAR cells. The Ct-UvrC was overexpressed with an N-terminal 6×His tag in E. coli BL21 (DE3) STAR cells. Cultures for UvrA, UvrB and UvrC were grown for 18 h at 18°C after induction with 0.4 mM isopropyl β-D-1-thiogalactopyranoside and were harvested by centrifugation. This was followed by the resuspension of cell pellets in lysis buffer that contained 25 mM HEPES (pH 7.5), 250 mM NaCl, 5% (v/v) glycerol, 10 mM β-mercaptoethanol, 10 mM imidazole, 1 mg/ml lysozyme, viscolase, and a mixture of protease inhibitors. Following incubation for 30 min on ice, the concentration of NaCl was increased to 500 mM. Cells lysis was performed by sonication, and the resulting cellular debris was removed by centrifugation at ~185,500×g (45 Ti rotor; Beckman Coulter). The recombinant proteins were primarily purified from the supernatant by affinity chromatography using a His-Trap column (GE Healthcare). The nickel column buffer (25 mM HEPES [pH 7.5], 500 mM NaCl, 5% (v/v) glycerol, 10 mM β-mercaptoethanol, and 10 mM imidazole) was used to pre-equilibrate the column. Following the loading of lysate, the column was washed consecutively with buffers that contained 10 and 100 mM imidazole, respectively. Ct-UvrA, Ct-UvrB and Ct-UvrC were eluted in nickel column buffer supplemented with 300 mM imidazole. The Ct-UvrA, Ct-UvrB and Ct-UvrC proteins with an N-terminal 6×His tag were concentrated and further purified by size exclusion chromatography (SEC; Hi-Load 16/600 Superdex-200; GE Healthcare) that was pre-equilibrated with SEC buffer (50 mM HEPES [pH 7.5], 250 mM NaCl, 5% (v/v) glycerol, and 1 mM dithiothreitol). Ct-UvrA and Ct-UvrB with an N-terminal 6×His-SUMO tag were subjected to SUMO protease treatment to remove the tag and dialysis at 4°C against nickel column buffer to reduce the imidazole concentration. The cleaved-off SUMO protein that was fused with the 6×His tag was removed on a His-Trap column (GE Healthcare). The cleaved proteins in the flow-through were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), concentrated, and further purified by SEC in SEC buffer. Eluted fractions were analyzed for purity by SDS-PAGE, and fractions that corresponded to pure protein were pooled and concentrated using an Amicon centrifugal filter device (Millipore), aliquoted, snap frozen, and stored separately at −80°C. Thermotoga maritima (Tm) UvrC was purified from the plasmid based on the pET28-SUMO vector23, using the same procedure as followed for the purification of 6×His-SUMO tagged Ct-UvrA and Ct-UvrB.

The K39A, K647A, Y62A, F66A, and Y62A/F66A variants of UvrA and the K44A variant of Ct-UvrB with point amino acid substitutions were generated by site-directed mutagenesis using the QuikChange method (Stratagene) or overlap extension polymerase chain reaction using KOD DNA polymerase (Novagen). The complete list of the primers used for cloning and mutagenesis is provided in Supplementary Data 1. The same procedure was followed for the purification of all point substitution variants as for the wild type proteins. Analysis of purified proteins used in this study is shown in Supplementary Fig. 1a.

Cryo-electron microscopy sample preparation and data collection
Samples for the cryo-EM studies of Ct-UvrA:DNA complexes in the presence/absence of nucleotide were reconstituted by pulling down the complex using 6×His-tagged Ct-UvrA. The oligonucleotides were ordered from Eurofins. The 50 bp flu-dT-DNA substrate with an internal fluorescein modification and mismatch opposite the modified base (F 50 variant)21,24,25 was prepared by annealing 50-mer EM1 5′-GACTACGTACTGTTACGGCTCCATCFGTACCGCAATCAGGCCAGATCTGC (where F is Fluorescein-dT) with 50-mer EM2 5′-GCAGATCTGG CCTGATTGCGGTACCGATGGAGCCGTAACAGTACGTAGTC. 6×His-Ct-UvrA (4.5 µM) and flu-dT-DNA (2.25 µM) in the presence of adenylyl-imidodiphosphate (AMPPNP; 200 µM) were incubated at room temperature in buffer P that contained 20 mM HEPES (pH 7.5), 5% (v/v) glycerol, 10 mM imidazole, 10 mM MgCl2, and 100 mM KCl for 15 min. The mixture was then cross-linked with glutaraldehyde (0.01%) for 15 min at room temperature and applied to Protino Ni-NTA agarose beads (Macherey-Nagel) that were pre-equilibrated with buffer P and incubated with beads for an additional 10 min at room temperature for immobilization of the Ct-UvrA and Ct-UvrA:DNA:AMPPNP complex via the 6×His tag on Ct-UvrA. Unbound protein and DNA were removed by four sequential washes (400 µl each) with wash buffer (20 mM HEPES [pH 7.5], 2% [v/v] glycerol, 10 mM MgCl2, and 150 mM NaCl). Protein that was bound to the Ni-NTA beads was eluted with buffer P that contained 250 mM imidazole (75 µl). AMPPNP was omitted in the mixture to reconstitute the sample in the absence of nucleotide or replaced with ADP for the Ct-UvrA:flu-dT-DNA:ADP sample.

Double-stranded DNA (dsDNA) with an abasic site was prepared by annealing 50-mer EM3 5′-GACTACGTACTGTTACGGCTCCATCXGTACCGCAATCAGGCCAGATCTGC (where X is the abasic site) and EM2. Unmodified dsDNA was prepared by annealing 50-mer EM4 5′-GACTACGTACTGTTACGGCTCCATCGGTACCGCAATCAGGCCAGATCTGC and EM2 oligonucleotides. The same purification procedure as described above was followed for reconstitution of the Ct-UvrA complex with DNA that contained an abasic site and unmodified DNA in the presence or absence of the nucleotide.

Initially, we prepared selected samples both with and without glutaraldehyde cross-linking. Although 2D classes and initial 3D reconstructions from both preparation methods showed very similar shapes, the micrographs from samples prepared without cross-linking had a significantly smaller number of good particles per micrograph, and the resulting reconstructions had a lower resolution. So, for the preparation of the samples reported herein, we included the cross-linking step.

The GraFix method56 was used for reconstitution of the Ct-UvrA:UvrB:flu-dT-DNA:AMPPNP complex, with a 10–30% (v/v) glycerol gradient in 20 mM HEPES (pH 7.5), 150 mM NaCl, and 10 mM MgCl2. For cross-linking the complex, 0.1% glutaraldehyde was supplemented in the 30% glycerol buffer. Ct-UvrA:UvrB:flu-dT-DNA:AMPPNP was reconstituted by mixing 3.6 µM flu-dT-DNA substrate, 7.2 µM UvrA, 14.4 µM UvrB, and 150 µM AMPPNP and loading on top of the glycerol gradient. The sample was ultracentrifuged at ~184,000×g for 18 h at 4°C in an SW 60 Ti rotor (Beckman Coulter), and 200 µl fractions were collected from the top to the bottom of the gradient. The fractions were analyzed by silver-stained SDS-PAGE. Fractions that corresponded to the complex were pooled and concentrated for vitrification with the simultaneous removal of glycerol.

The Ct-UvrA, Ct-UvrA:DNA and Ct-UvrA:UvrB:DNA samples (3 µl) were vitrified in liquid ethane on glow-discharged Quantifoil 2/1 mesh 200 Au, C-flat 2/1 mesh 200 Cu, and customized Graphene oxide-coated Quantifoil 2/1 mesh 200 Cu grids, respectively, using an FEI Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 95% humidity, and with a 4 s blot time for Quantifoil and C-flat grids and 30 s wait time and 1.5 s blot time for Graphene oxide-coated grids. A Glacios electron microscope (Thermo Fisher Scientific) with a Falcon 3EC camera at 200 kV (Center of New Technologies, University of Warsaw) was used for initial screening of the grids. The final datasets for all the samples were collected on a Titan Krios G3i electron microscope (Thermo Fisher Scientific) that was equipped with a BioQuantum energy filter (with 20 eV energy slit) and K3 camera (Gatan) at 300 kV at the SOLARIS National Synchrotron Radiation Center (Krakow, Poland). Data were collected as movies in counting mode with a physical pixel size of 0.84 Å (nominal magnification of 105,000×), 50 µm C2 aperture, and retracted objective aperture. The total dose (fractionated into 40 frames) was ~40 e/Å2, and the dose rate was ~16 e/pixel/s (for details, see Supplementary Table 1).

Cryo-electron microscopy data processing and interpretation
Cryo-electron microscopy images were processed with RELION-4.0-5.057 and cryoSPARC 4.4-4.558 (Supplementary Figs. 2–13 and Supplementary Methods).

2-Aminopurine fluorescence measurements
Three DNA substrates (AP-1, AP-2, and AP-3) were used for the assay. AP-1 substrate, with flu-dT and 2-Aminopurine in the center, was generated by annealing EM1 with AP-a 5′-GCAGATCTGGCCTGATTGCGGTAC(P)GATGGAGCCGTAACAGTACGTAGTC (where P is 2-Aminopurine). To generate AP-2, with 2-Aminopurine in the center in one strand and no modification in the other strand, AP-b 5′-GACTACGTACTGTTACGGCTCCATCTGTACCGCAATCAGGCCAGATCTGC was annealed with AP-a oligonucleotide. For AP-3 substrate, with flu-dT in the center and 2-Aminopurine near the end, EM1 oligonucleotide was annealed with AP-c 5′-GCAGATCTGGCCTGATTGCGGTACCGATGGAGCCGTAACAGT(P)CGTAGTC (where P is 2-Aminopurine). DNA (2.5 µM) was mixed with Ct-UvrA (10 µM) in the presence or absence of 500 µM AMPPNP. The assay was performed in 20 mM HEPES (pH 7.5), 100 mM KCl, 5% (v/v) glycerol, 5 mM MgCl2, and 1 mM DTT. Fluorescence emission spectra were recorded using a Jasco FP-8300 spectrofluorometer that was connected to a water bath to maintain a 37°C temperature inside the cuvette. The excitation wavelength was set at 320 nm, and emission spectra were recorded at 340–440 nm. Both the excitation and emission slits were set at a width of 5 nm. Fluorescence was measured using a quartz cuvette with a 10 mm path length. The results were corrected for background fluorescence by subtracting the spectrum of the buffer. For each experiment, measurements were accumulated from three scans. Finally, percentage increases in fluorescence at a 370 nm wavelength were plotted. Three independent experiments were performed for each reaction.

UvrB loading experiments
Interactions of Ct-UvrA and Ct-UvrB with flu-dT and unmodified DNA (DNA substrates used for cryo-EM sample preparation) in the presence of AMPPNP, ATP and ADP and absence of nucleotide, were studied by the pull-down of Ct-UvrA (without His-tag) and DNA with 6×His-tagged Ct-UvrB. For the reaction, Ct-UvrA (0.75 μM) was incubated with DNA (0.375 μM) and nucleotide (100 μM) at room temperature for 10 min in buffer S that contained 20 mM HEPES (pH 7.5), 5% (v/v) glycerol, 5 mM imidazole, 10 mM MgCl2, and 100 mM KCl. 6×His-Ct-UvrB (0.75 μM) was added to the mixture and incubated at room temperature for an additional 30 min. The reaction mixture was applied to Protino Ni-NTA agarose beads (Macherey-Nagel) that were pre-equilibrated with buffer S and incubated with beads for an additional 10 min. Unbound proteins were removed by two washes (200 µl each) with wash buffer A (20 mM HEPES [pH 7.5], 5% [v/v] glycerol, 10 mM MgCl2, and 150 mM NaCl) and three washes with wash buffer B (20 mM HEPES [pH 7.5], 5% [v/v] glycerol, 10 mM MgCl2, and 1 M NaCl). Proteins that were immobilized on Ni-NTA beads were eluted with buffer S supplemented with 250 mM imidazole (100 µl). Three independent experiments were performed for each reaction. Proteins in the elution, washes, and flow-through were analyzed by SDS-PAGE that was stained with Coomassie Brilliant Blue R-250. The DNA was analyzed by 20% TBE-urea-PAGE that was stained with ethidium bromide.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study the authors have used cryo-EM and biochemical methods to study the role of ATP hydrolysis in the binding of UvrA and UvrA-UvrB to a DNA duplex containing unmodified DNA, a fluorescein-dT (Fl-dT) moiety or an abasic site. UvrA forms a dimer on DNA and thus has four ATP binding sites per complex. The paper nicely details complexes of UvrA without a nucleotide, AMP-PNP and ADP on non-damaged DNA and DNA with a Fl-dT moiety. The authors report several conformational states of UvrA and DNA as it engages damage in the absence and presence of ADP or AMP-PNP. Most notable a bend of about 20 degrees and insertion of structural wedge into the DNA during damage search. They confirm the opening of the DNA using a fluorescence assay and validation of several important amino acids at the DNA –UvrA wedge interface. The authors are also aware of and carefully point out the fact that UvrA binds to DNA ends and some of the structures could be bridges between two end binding events which due to low density cannot be fully interpreted. Work-up and discussion of these structures were therefore not pursued.\nOverall the results presented in this study are important and novel. The authors have found that bindng of AMP-PNP produces a profound alteration in the conformation of UvrA binding to DNA. It would appear that this AMP-PNP bound UvrA-DNA complex is similar for both damaged DNA and non-damaged DNA, and thus provides important information on the search process, but not the actual final damage recognition process. UvrA ATPase is stimulated by DNA damage and a recent study by Barnett and Kad (FASEB FASEB J. 2019 Jan;33(1):763-769; which needs to be cited and discussed) suggest that actual damage engagement can lead to allosteric effects on the ATPase activity of one of the two sites. Thus, the AMP-PNP trapped complex is interesting, but not fully engaging the DNA damage. Unfortunately, as the authors clearly cite UvrA’s specificity increases several orders of magnitude for damaged sites in the presence of ATP. One can appreciate that adding ATP to the UvrA-DNA complex will result in ATP hydrolysis and subsequent ADP binding. However, the authors fail to fully appreciate that specificity for DNA damage is completely abrogated (and do not cite the pertinent literation) in the presence of poorly hydrozylable ATP-gamma-S (Sancar, Grossman, Goosen laboratories) and therefore probably AMP-PNP. Clearly ATP hydrolysis is essential for UvrB loading at damaged sites, as confirmed by multiple laboratories, thus the UvrA-UvrB-DNA complex while quite interesting and relevant is only the pathway to damage discrimination and not showing UvrB engagement (see Kraithong et al, DNA Repair 2021). Thus, while this manuscript provides impressive structural work-up of UvrA bound to DNA in several different states is very exciting and important, the final conclusions need to be carefully tempered by a vast of literature which is only partially cited. It is not clear that these studies as currently described are actually providing, as their title implies, “mechanism of ATP-dependent DNA damage recognition by UvrA.” The authors should consider the following points:\n1. Please provide a SDS gel of all the proteins used in this study.\n2. The authors are encouraged to move a large amount of the materials methods to the supplement (which runs for 19 pages), and move supplemental results, “Configuration of ATPase active site and their coordination (lines 50-105), and section “The mechanism of coupling DNA binding and ATPase activity” (lines 107-139) into the main text as these studies have great relevance to the title of the manuscript.\n3. Line 588, the authors state that an abasic site “… is not typically recognized by bacterial NER”. This is a vague statement. Snowden and Van Houten( J Mol Biol. 1991 Jul 5;220(1):19-33), showed that while UvrA binds avidly to DNA containing an abasic as compared to nondamaged DNA, UvrB is poorly loaded and thus this lesion is not incised efficiently. Please add this reference.\n4. Line 686, the authors note large structural differences between UvrA binding to the Fl-dT in the absence and presence of AMP-PNP. Based on my discussion above this wedge insertion and bending of DNA and subsequent untwisting of the strands, is probably an important mode to damage recognition. However since non-hydrolyzable ATP completely abolishes UvrA specificity (see paper by the Sancar JBC 1988, 263(32):16553-60 for example) this large structural change is part of the recognition process, but not the final UvrA-DNA damage complex that then hands off to UvrB. Thus, their nicely word conclusion on lines 784-785 is probably correct, but the authors are encouraged to provide a clear statement that this wedge complex is not the entire engagement of the DNA damage. If the Barrnett & Kad paper is correct, then upon UvrA engaging the lesion it is only then that the second ATPase site is activated. The authors must put their results into the large context of the work-up of the ATPase site variants as reported in the supplement, and discuss the implications of the Kad paper.\n5. It might be good to show a superpostion the two structures discussed on lines 863-864 in the supplement.\n6. The complexes of UvrA-UvrB with DNA are important, but need to be presented cautiously within the context that hand off the lesion to UvrB has not occurred since ATP hydrolysis is required for beta-hairpin engagement at the damaged site. While data in Figure 5 panels C and D would appear to provide information UvrB loading these results are inconsistent with a series of papers from the Goosen, Grossman, Sancar, and Van Houten laboratories, as well as the recent paper by see Kraithong et al, DNA Repair 2021. Thus their sentence on lines 899-900, about UvrB loading needs to explicitly state that in their experiments they did not observe UvrB loading since ATP hydrolysis is required. The authors need to be cautious with their interpretation of the biochemical pull down experiments.\n7. The discussion needs to include citations to older literature demonstrating that non-hydrolyzable ATP analogs increase UvrA binding to nondamaged DNA and thus abrogate all damage specificity. That the authors have shown the insertion of the wedge into non-damaged in the presence of AMP-PNP, they have actually solved a long standing problem in the field of why non-hydrolyzable ATP analogs block specificity. They have trapped an intermediate with AMP-PNP, but hydrolysis will be necessary to show exact engagement to the lesion, perhaps flooding UvrA-DNA complexes with ATP and time resolved structures might show the proper engagement with DNA damage.", "reviewer_2": "Overall, this is a welcome study looking at the real complexes of UvrA, DNA and UvrB found only by cryo-EM. The number of structures investigated and the conditions permutated through make this a really powerful study. The conclusions are very interesting and will stimulate experiments to test some of these aspects of mechanism, therefore this will be of wide interest within and outside the field.\nThere are a few criticisms I would make in no particular order of seriousness:\n(1) Where is the supporting evidence that Ct NER follows the same biochemistry as the other species studied thus far. I understand it would be unlikely that there would be large differences, but given a lot of the detail in this study is fairly subtle at least some data showing the repair of DNA in mesothermic conditions can be seen.\n(2) Following on, given you are using room temperatures with a thermophilic enzyme, what are the expectations regarding the structure of the DNA when hotter, i.e. the working temp for this enzyme? I would really value a discussion of this topic, here modeling would be welcome.\n(3) The final conclusions suggest that ATP is not important for damage recognition, but it should be worth noting that ATP hydrolysis is necessary for UvrB loading. What would be really supportive of these suggestions would be a comparison of UvrB loading with and without ATP, or even using their own expts (pull down) showing different lesions do or do not require ATP.\n(4) Ref 24 is a mess!\n(5) Line 954 reference to 57 is incorrect – that study was in vivo, do the authors mean to use ref 59 here?\n(6) Line 934: An interesting observation here is that end-binding occurs with undamaged DNA. This suggests that UvrA would have transient interactions along the DNA looking for damage, until it finds something that is more stable, such as the Flu-dT, what is the relative preference for damage in the middle of the DNA vs the end? This should be inherent with the distribution of particles I would have thought?\n(7) Line 928: I think that this conclusion needs to be supported by data. There are a lot of differences between the 2xFlu-dT in a different species and this. Ideally, you would need to have the structure of two Fl-dTs in Ct. This is not an experiment I would want to see, rather a modulation of the statement!\n(8) Line 909, but really a concept that sits throughout the study: Given that mixed nucleotides are the most likely occupiers of the ATPase site (see the studies from the Hignorani group) hpw relevant is the saturation with a single nucleotide species?\n(9) I was a little surprised that UvrB loaded so strongly, such that it could resist 1M NaCl, the current hypothesis is that UvrB needs an ATP turnover to engage the b-hairpin, so how does that reconcile with the AMPPNP observations here? A wider discussion of this point would be really welcome, especially since the AMPPNP data collected affects both UvrB and UvrA.\n(10) Line 821: DO the authors agree that an alternative hypothesis to their results on the lysine ATPase knockout mutants could be that AMPPNP needs to be in both distal and proximal at the same time?\n(11) Have the authors any idea about UvrB loading with the Y62 and N270 area mutations? My guess would be that UvrB couldn’t load, do they have any data (e.g. pull down) that could add to that part of their (very interesting) story?\n(12) Is it possible that endogenous UvrA or UvrB could complex with the Ct orthologues?\n(13) Could the authors explain why cross-linking was needed? Also, could they make it clearer in the results that these structures were cross-linked, rather than only mentioning it in the methods.", "reviewer_3": "Nucleotide excision repair (NER) is a critical pathway for the maintenance of genome stability in response to various types of DNA damage. A key step in this pathway is the recognition of damage and subsequent recruitment of downstream effector proteins. In bacteria, DNA damage is recognized by the ATPase UvrA, which initiates the NER pathway by recruiting UvrB. The manuscript by Nirwal et al. presents a detailed structural analysis of DNA-engagement by UvrA and recruitment of UvrB, giving important insights into key steps in the bacterial NER pathway. The manuscript is built around a series of cryogenic electron microscopy (cryo-EM) structures (14 deposited PDBs) of UvrA +/- nucleotides, +/- DNA and +/- UvrB that illustrate distinct UvrA/DNA conformational states. The cryo-EM work is solid and the high-resolution of the structures (albeit with reduced resolution in the DNA) allow the authors to propose a mechanism for how DNA damage is recognized by UvrA.\nThe cryo-EM data represents a thorough, detailed analysis of UvrA interactions with DNA under a range of mechanistically relevant conditions, the manuscript, figures and accompanying figure legends are clear and include important acknowledgement of certain limitations of the data (e.g. weak DNA density in some regions of the cryo-EM maps; possible ‘end-binding’). The data is well supported by a comprehensive ‘Materials and Methods’ section and supplementary materials (incl. an excellent movie summarizing the structural states observed in the EM data). Overall, this is an impressive piece of work and it is evident that the authors have taken great care and consideration in preparing the manuscript. I only have a few relatively minor points that I hope will help to further improve the manuscript.\n1. The structures and accompanying data offer important insights into the interactions between UvrA, DNA and UvrB and how different functional states relate to nucleotide binding. These structures, viewed in the context of previously published data allow the authors to speculate about the mechanism by which UvrA specifically recognizes sites of DNA damage. However, the data presented here, as it stands, does not demonstrate specific binding to (or preference for) damaged DNA, or distinct structural features that might explain preferential binding to damaged DNA. On the contrary, the structures and biochemical assays (e.g. UvrB loading) appear to show little difference between unmodified and modified DNA, making many of the key statements of conclusions (incl. the title) difficult to support. For example, the statement “our results present a complete description of a unique mechanism of DNA damage detection by …” overstates what can be concluded from the data presented. This is not to diminish the quality of the work, the value of the structures or the merit of publication, but the conclusions should be reasonable given the data presented and I would recommend careful consideration of what can be concluded.\n2. The main distinguishing feature in the data between damaged and undamaged DNA appears to be the presence of ATP-bound conformation 2, which was identified for the abasic and unmodified DNA but not for flu-dT DNA. Whilst noted, little comment is made on this observation. Related to this, in the EM map for ATP-bound conformation 2 with unmodified DNA (EMD-51669), density appears to be present for the flipped base, but this is not built into the current models (see attached images). This density is absent in the DNA containing an abasic site (albeit the sharpened map is noisier). Is it possible that recognition of certain types of DNA damage is related to an inability to form ATP-bound conformation 2 (potentially with consequences for ATP hydrolysis and substrate release)?\n3. Related to the above, lines 588-589 are not clear – is the abasic DNA used in this study a target for the NER system studied here? Does this substrate represent a genuine target for UvrA or a ‘control’ non-target sequence, like the unmodified DNA?\n4. C2 symmetry is applied in the processing of all EM structures, could the authors please confirm that the processing in C1 has been investigated, and the application of symmetry does not influence the maps, in particular the DNA density?\n5. In the materials and methods, the authors mention performing a 3DFlex analysis comparing nucleotide bound and free states, but this data is currently not included in the paper. This would be interesting to see as a supplement, to compare the relative flexibility of different states.\n6. In the 2-aminopurine base flipping experiments, the authors plot the % change in fluorescence, rather than absolute values. They suggest that the larger increase in fluorescence for the unmodified DNA may be because there is “no pre-existing perturbations of base pairing in this DNA”. Is the baseline fluorescence significantly higher in the flu-dT DNA because of perturbed basepairing, supporting this proposal, or is it possible that the ‘unmodified’ DNA is more amenable to flipping and this is important for distinguishing between undamaged and damaged DNA. For example, if ATP-bound state 2 cannot be formed by large adducts on a base, base flipping might more difficult on this substrate despite the fact that base pairing is disrupted by the damage.\n7. Figure 4A and B could be significantly improved to better display the wedge element that causes base flipping. The current figure is very hard to interpret and this key feature of the structures should be clearly highlighted.\n8. In the discussion the authors mention UvrA having a ‘moderate specificity for modified DNA’ (Lines 919 and 942), it would help the reader to be more specific."}, "author_response": "Point-by-point response\nNirwal S. et al., Structural snapshots of the mechanism of ATP-dependent DNA damage\nrecognition by UvrA.\nBelow we provide a point-by-point response with a description of the changes introduced in\nthe manuscript. We found the comments of the Reviewers constructive and helpful and we\nbelieve that they allowed us improve the manuscript.\nReviewer #1 (Remarks to the Author):\nIn this study the authors have used cryo-EM and biochemical methods to study the role of\nATP hydrolysis in the binding of UvrA and UvrA-UvrB to a DNA duplex containing unmodified\nDNA, a fluorescein-dT (Fl-dT) moiety or an abasic site. UvrA forms a dimer on DNA and thus\nhas four ATP binding sites per complex. The paper nicely details complexes of UvrA without\na nucleotide, AMP-PNP and ADP on non-damaged DNA and DNA with a Fl-dT moiety. The\nauthors report several conformational states of UvrA and DNA as it engages damage in the\nabsence and presence of ADP or AMP-PNP. Most notable a bend of about 20 degrees and\ninsertion of structural wedge into the DNA during damage search. They confirm the opening\nof the DNA using a fluorescence assay and validation of several important amino acids at the\nDNA –UvrA wedge interface. The authors are also aware of and carefully point out the fact\nthat UvrA binds to DNA ends and some of the structures could be bridges between two end\nbinding events which due to low density cannot be fully interpreted. Work-up and discussion\nof these structures were therefore not pursued. Overall the results presented in this study are\nimportant and novel.\nWe are very glad to hear that this Reviewer considers our work is important and novel.\nThe authors have found that bindng of AMP-PNP produces a profound alteration in the\nconformation of UvrA binding to DNA. It would appear that this AMP-PNP bound UvrA-DNA\ncomplex is similar for both damaged DNA and non-damaged DNA, and thus provides\nimportant information on the search process, but not the actual final damage recognition\nprocess. UvrA ATPase is stimulated by DNA damage and a recent study by Barnett and Kad\n(FASEB FASEB J. 2019 Jan;33(1):763-769; which needs to be cited and discussed) suggest\nthat actual damage engagement can lead to allosteric effects on the ATPase activity of one of\nthe two sites. Thus, the AMP-PNP trapped complex is interesting, but not fully engaging the\nDNA damage.\nWe are sorry for the unintentional omission of the reference to an important paper by Barnett\nand Kad which must have happened accidentally during the editing of the manuscript. There\nwas a sentence referring to the paper by Barnett and Kad in the submitted version of the text\nbut the reference was missing. We edited this sentence slightly to “It was also shown that the\nresidence time of the protein on UV-damaged DNA was longer than on undamaged DNA in\nthe presence of ATP, but with ATPγS, UvrA stably bound both types of DNA” on page 21. We\nnow also provide the proper reference. In fact, this important paper is fully in line with our\ninterpretation of our structural data. We postulate that cycles of ATP hydrolysis leading to\nconformational changes of UvrA dimer result in faster dissociation of the protein from less\nmalleable undamaged DNA. To stress this we added an additional sentence “The same work\npostulated that it is the step between ATP binding and its hydrolysis to ADP which serves to\ndetect the damage. This is in agreement with our interpretation that conformational changes\ndriven by ATP hydrolysis are key for damage recognition. These findings imply that\ntransitions between states play a key role and a kinetic damage discrimination mechanism is\nused by UvrA.”\nrecognition process. These are snapshots of a dynamic process of lesion localization which\ninvolves kinetic proofreading and relies on differences in residence times. To acknowledge\nthis we added the following paragraph at the end of the Discussion:\n“The damage recognition process by bacterial NER is clearly dynamic. Our structural data\nprovide snapshots of key steps this process. Its full understanding will require additional\nstudies guided by our structural data which will include time-resolved cryo-EM, single-\nmolecule biophysics and molecular dynamics simulations. Together with the structure\npresented herein they will provide a detailed comprehensive picture of damage recognition\nby bacterial NER.”\nUnfortunately, as the authors clearly cite UvrA’s specificity increases several orders of\nmagnitude for damaged sites in the presence of ATP. One can appreciate that adding ATP to\nthe UvrA-DNA complex will result in ATP hydrolysis and subsequent ADP binding. However,\nthe authors fail to fully appreciate that specificity for DNA damage is completely abrogated\n(and do not cite the pertinent literation) in the presence of poorly hydrozylable ATP-gamma-S\n(Sancar, Grossman, Goosen laboratories) and therefore probably AMP-PNP. Clearly ATP\nhydrolysis is essential for UvrB loading at damaged sites, as confirmed by multiple\nlaboratories, thus the UvrA-UvrB-DNA complex while quite interesting and relevant is only\nthe pathway to damage discrimination and not showing UvrB engagement (see Kraithong et\nal, DNA Repair 2021). Thus, while this manuscript provides impressive structural work-up of\nUvrA bound to DNA in several different states is very exciting and important, the final\nconclusions need to be carefully tempered by a vast of literature which is only partially cited.\nIt is not clear that these studies as currently described are actually providing, as their title\nimplies, “mechanism of ATP-dependent DNA damage recognition by UvrA.”\nIndeed, the specific recognition of DNA modification requires ATP. We now present pull-down\nexperiments performed in the presence of ATP to show this also in our system. Please see\nthe response to point 4. below. Please also see the response to point 6. below regarding the\nengagement of UvrB at the lesion.\nA similar comment about tempering our conclusions was made by Referee 3. As pointed out\nin the response above, we provide snapshots of the process and additional studies will be\nneeded to fully characterize the mechanism. We carefully went through the text and softened\nthe language. We also changed the title to:\n“Structural snapshots of the mechanism of ATP-dependent DNA damage recognition by\nUvrA”\nAs mentioned above, we also added a sentence at the end of the Discussion section starting\nwith\n“The damage recognition process by bacterial NER is clearly dynamic...”\nThe authors should consider the following points:\n1. Please provide a SDS gel of all the proteins used in this study.\nWe have added an SDS-PAGE analysis of the proteins used in this study as Supplementary\nFig. 1a. All subsequent figures have been renumbered.\n2. The authors are encouraged to move a large amount of the materials methods to the\n2\nsupplement (which runs for 19 pages), and move supplemental results, “Configuration of\nATPase active site and their coordination (lines 50-105), and section “The mechanism of\ncoupling DNA binding and ATPase activity” (lines 107-139) into the main text as these studies\nhave great relevance to the title of the manuscript.\nThe manuscript has been edited as suggested. The appropriate sections have been moved\nto the main text and figures renumbered accordingly.\n3. Line 588, the authors state that an abasic site “… is not typically recognized by bacterial\nNER”. This is a vague statement. Snowden and Van Houten( J Mol Biol. 1991 Jul\n5;220(1):19-33), showed that while UvrA binds avidly to DNA containing an abasic as\ncompared to nondamaged DNA, UvrB is poorly loaded and thus this lesion is not incised\nefficiently. Please add this reference.\nThe manuscript has been edited as suggested and relevant reference added.\n4. Line 686, the authors note large structural differences between UvrA binding to the Fl-dT in\nthe absence and presence of AMP-PNP. Based on my discussion above this wedge insertion\nand bending of DNA and subsequent untwisting of the strands, is probably an important\nmode to damage recognition. However since non-hydrolyzable ATP completely abolishes\nUvrA specificity (see paper by the Sancar JBC 1988, 263(32):16553-60 for example) this\nlarge structural change is part of the recognition process, but not the final UvrA-DNA damage\ncomplex that then hands off to UvrB. Thus, their nicely word conclusion on lines 784-785 is\nprobably correct, but the authors are encouraged to provide a clear statement that this\nwedge complex is not the entire engagement of the DNA damage. If the Barrnett & Kad\npaper is correct, then upon UvrA engaging the lesion it is only then that the second ATPase\nsite is activated. The authors must put their results into the large context of the work-up of the\nATPase site variants as reported in the supplement, and discuss the implications of the Kad\npaper.\nThe text page 11 was edited to: “The likely purpose of this insertion is to actively probe\nstability of the DNA, particularly the strength of base pairing, which would be reduced for a\ndamaged duplex. However, it is possible that this complex does not represent the entire\nengagement of the DNA damage.”\nPrompted by this comment and other comments of the Reviewers, we performed pull-down\nexperiments in the presence of ATP. They are shown in updated Fig. 7c, d, e, f. These results\nshowed that the presence of ATP promoted preferential loading of UvrB on modified versus\nunmodified DNA. We also tested two variants of UvrA with substitution of the wedge residue\nY62 and a nearby residue N270. For Y62A substitution the loading of UvrB on modified DNA\nin the presence of ATP was compromised and specificity for modified DNA was lost. This\nagrees with the suggestion of the Referee that the wedge is important for ATP-dependent\ndamage discrimination. N270A showed loading indistinguishable from the wild-type UvrA (not\nshown).\nThe coordination of the two ATPase active sites is a very complex process. Our structural\ndata clearly show that only when both active site are occupied by AMPPNP, UvrA can adopt\nATP-bound conformation 1 or 2. Moreover, as described below, our preliminary data show\nthat mixed states of UvrA with ATP bound at one active site and the other one hydrolyzing\nATP are not efficient in damage discrimination. So, the two active sites cooperate in lesion\ndetection.\nbind and hydrolyze ATP will require careful MD simulations and single-molecule experiments\nguided by our structural data. The static pictures from our structural data are not sufficient to\nfully explain the dynamic nature of this coordination.\n5. It might be good to show a superpostion the two structures discussed on lines 863-864 in\nthe supplement.\nThe appropriate panel with comparison of the two structures has been added to\nSupplementary Figure 19.\n6. The complexes of UvrA-UvrB with DNA are important, but need to be presented cautiously\nwithin the context that hand off the lesion to UvrB has not occurred since ATP hydrolysis is\nrequired for beta-hairpin engagement at the damaged site. While data in Figure 5 panels C\nand D would appear to provide information UvrB loading these results are inconsistent with a\nseries of papers from the Goosen, Grossman, Sancar, and Van Houten laboratories, as well\nas the recent paper by see Kraithong et al, DNA Repair 2021. Thus their sentence on lines\n899-900, about UvrB loading needs to explicitly state that in their experiments they did not\nobserve UvrB loading since ATP hydrolysis is required. The authors need to be cautious with\ntheir interpretation of the biochemical pull down experiments.\nThis is an important consideration and this was also raised by Reviewer #2.\nOne point to clarify is the definition of UvrB loading. There are two possible states. In one\nstate the β-hairpin can clamp a DNA strand away from the DNA damage. We assume this\nconfiguration arises right after UvrB is loaded on the DNA by UvrA - the state visualized by\nour UvrA:UvrB:DNA complex structure. In the structure β-hairpin clearly clamps the DNA. To\nvisualize this we added insets with close-ups of the β-hairpin and cryo-EM potential maps to\nFig. 7a, b (previously 5A, B).\nThe other state is the one in which the hairpin is locked at the site of DNA modification. Our\nUvrA:UvrB complex does not represent this state. As pointed out by the referee this requires\nATP hydrolysis. We assume that this hydrolysis is required for the UvrB to reach the lesion\nby performing ATP-dependent translocation on DNA (see PMID: 31739207) So, the UvrB\nloading according to the second definition (engagement of the β-hairpin at the modification)\nclearly requires ATP hydrolysis. We would therefore argue that our results and their\ninterpretation is in agreement with the available data, in particular with the paper of Kraithong\net al. (PMID: 33302090, Fig. R1). This information has been added to the discussion on Page\n22:\n“Our UvrA:UvrB:DNA structure was determined in the presence of AMPPNP, so the formation\nof a complex with UvrB clamping one of the DNA strands was independent of ATP hydrolysis.\nMoreover, our pull-down experiments in the presence of AMPPNP for wild-type UvrB and for\nATPase-deficient variant of UvrB (K44A) showed the formation of high salt concentration-\nresistant UvrB:DNA complex. We assume that this very stable complex involves clamping of\nthe DNA strand, as shown by our UvrA:UvrB:DNA complex structure. So, we do observe the\nATP hydrolysis-independent formation of UvrB:DNA complex. In agreement with this, several\nworks showed that in absence of ATP or for K45A mutant of UvrB (E. coli and G.\nstearothermophilus numbering), which is unable to hydrolyze ATP, UvrA:UvrB:DNA complex\nis formed (PMID: 16282327, 2671996, 33302090, 20227373).”\nbox marks the state we postulate was captured in our Ct-UvrA:UvrB:DNA complex structure.\n----\nHere is the summary of the papers that are consistent with our findings. They are now all\ncited in our manuscript. We give slightly reworded quotes form the papers:\nSeeley, T. W. & Grossman, L. Mutations in the Escherichia coli UvrB ATPase motif\ncompromise excision repair capacity. Proc. Natl. Acad Sci. USA 86, 6577-6581(1989).\nLys45 of UvrB Is essential for UV resistance. All plasmids with Lys45 mutation, failed entirely\nto enhance UV survival and were indistinguishable from cells carrying either pUC19 vector or\nno plasmid.\nUvrB K45A Interacts with UvrA on DNA: Under conditions in which wild-type UvrB and N51A\nprotein formed nucleoprotein complexes on UV-irradiated DNA, the K45A mutant protein also\nformed complexes, although with a reduced yield.\nKraithong, T. et al. Real-time investigation of the roles of ATP hydrolysis by UvrA and UvrB\nduring DNA damage recognition in nucleotide excision repair. DNA Repair 97, 103024\n(2021).\nATP hydrolysis by UvrB is essential for the formation of UvrB-DNA pre-incision complex but\nis not required for binding between UvrAB and damaged DNA.\nMalta, E., Moolenaar, G. F. & Goosen, N. Base Flipping in Nucleotide Excision Repair.\nJournal of Biological Chemistry 281, 2184–2194 (2006).\nK45A mutation resulted in a protein still capable of interacting with UvrA but unable to\nhydrolyze ATP. When UvrA and UvrB were mixed with DNA and the formation of complexes\nwas monitored by Electrophoretic mobility shift assay, UvrB(K45A) formed UvrA-UvrB-DNA\ncomplex, but no UvrB-DNA complexes. In addition, for this mutant of UvrB, there is no\ndetectable incision by UvrC or Cho.\nJournal of Biological Chemistry 265, 7158–7165 (1990).\nMutation at lysine 45 in the ATPase motif of UvrB specifically inhibits a key step in nucleotide\nexcision repair involving the UvrB ATPase-dependent translocation of nucleoprotein\ncomplexes from undamaged to damaged DNA sites.\nKad, N. M., Wang, H., Kennedy, G. G., Warshaw, D. M. & Van Houten, B. Collaborative\nDynamic DNA Scanning by Nucleotide Excision Repair Proteins Investigated by Single-\nMolecule Imaging of Quantum-Dot-Labeled Proteins. Molecular Cell 37, 702–713 (2010).\nATP was not required for the formation of dual-labeled UvrAB complexes or their subsequent\nassociation with the DNA. Thus, in contrast to bulk assays where the end points are\nproductive, binding to a site-specific lesion in a DNA substrate, it was found that ATP was\nunnecessary to form a UvrAB complex that is capable of binding to DNA. (single-molecule\nimaging of quantum-dot-labeled proteins).\n----\nWe cannot exclude the possibility that the loading of β-hairpin on the DNA is possible in our\nsystem because we use synthetic DNA duplexes with blunt ends which can serve as entry\npoints for UvrB. However, our preliminary results showed that UvrB can also be loaded on\nplasmid DNA which makes this possibility less likely.\n7. The discussion needs to include citations to older literature demonstrating that non-\nhydrolyzable ATP analogs increase UvrA binding to nondamaged DNA and thus abrogate all\ndamage specificity. That the authors have shown the insertion of the wedge into non-\ndamaged in the presence of AMP-PNP, they have actually solved a long standing problem in\nthe field of why non-hydrolyzable ATP analogs block specificity. They have trapped an\nintermediate with AMP-PNP, but hydrolysis will be necessary to show exact engagement to\nthe lesion, perhaps flooding UvrA-DNA complexes with ATP and time resolved structures\nmight show the proper engagement with DNA damage.\nThis is a helpful comment. Indeed, our new pull-down experiments in the presence of ATP\nshow damage-specific UvrB loading on the DNA. We have added a sentence on page 19:\n“This observation is in agreement with the previous findings that showed that non-\nhydrolyzable analogs of ATP attenuate the specificity of UvrA for damaged DNA” with\nrelevant references.\n----\nHere is a summary of previous data, including older papers:\nVan Houten, B., Gamper, H., Hearst, J. E. & Sancar, A. Analysis of sequential steps of\nnucleotide excision repair in Escherichia coli using synthetic substrates containing single\npsoralen adducts. Journal of Biological Chemistry 263, 16553–16560 (1988).\nAdenosine 5’-0-(thiotriphosphate) increases nonspecific binding and completely abolishes\nthe UvrA footprint (shown by DNA footprinting)\nBarnett, J. T. & Kad, N. M. Understanding the coupling between DNA damage detection and\nUvrA’s ATPase using bulk and single molecule kinetics. FASEB J 33, 763–769 (2019).\nDetachment rate in presence of ATP is higher for undamaged DNA.\nSeeberg, E. & Steinum, A. L. Purification and properties of the uvrA protein from Escherichia\ncoli. Proc Natl Acad Sci U S A 79, 988–92 (1982).\nATP[yS] abolishes the preferential binding to UV-irradiated DNA (shown by filter-binding\nassay)\nMazur, S. J. & Grossman, L. Dimerization of Escherichia coli UvrA and its binding to\nundamaged and ultraviolet light damaged DNA. Biochemistry 30, 4432–43 (1991).\nIn the presence of ATPyS, stable complexes are formed between UvrA and undamaged DNA\nor with UV-irradiated DNA (shown by gel mobility shift assay).\nWagner, K., Moolenaar, G., van Noort, J. & Goosen, N. Single-molecule analysis reveals two\nseparate DNA-binding domains in the Escherichia coli UvrA dimer. Nucleic Acids Res 37,\n1962–72 (2009).\nAFM data show that the binding affinity for DNA damage is significantly reduced in the\npresence of ATPγS.\n----\nPrompted by this comment we also analyzed the buried surface area in the representative\nstructures of the three conformations observed in our structural work. The values are as\nfollows:\nCt-UvrA:flu-dT (basal conformation): 1598.7 Å2 (1040.5 Å2without DNA-binding domains)\nCt-UvrA:abasic:AMPPNP (ATP-bound conformation 1): 2503.1 Å2\nCt-UvrA:abasic:AMPPNP (ATP-bound conformation 2): 2589.9 Å2\nThese values clearly show that in ATP-bound conformations stabilized by non-hydrolyzable\nanalogs the buried surface area is much larger explaining the high affinity of UvrA for DNA in\nthe presence of these analogs. This information has been added to the text on page 12.\nTime-resolved cryo-EM is time- and resource-consuming and is beyond the scope of this\nstudy, but should be definitely undertaken in the future (we now state this in the text). As we\npoint out, our structures also form a very valuable basis for the studies using MD simulations\nand single-molecule experiments.\nReviewer #2 (Remarks to the Author):\nOverall, this is a welcome study looking at the real complexes of UvrA, DNA and UvrB found\nonly by cryo-EM. The number of structures investigated and the conditions permutated\nthrough make this a really powerful study. The conclusions are very interesting and will\nstimulate experiments to test some of these aspects of mechanism, therefore this will be of\nwide interest within and outside the field.\nWe are very glad to hear this positive assessment.\n(1) Where is the supporting evidence that Ct NER follows the same biochemistry as the other\nspecies studied thus far. I understand it would be unlikely that there would be large\ndifferences, but given a lot of the detail in this study is fairly subtle at least some data\nshowing the repair of DNA in mesothermic conditions can be seen.\nWe performed DNA incision assays with Ct-UvrA:UvrB:UvrC combination and flu-dT-DNA. In\nthis setup UvrC could only introduce one incision. However, when Ct-UvrC was replaced by\nTm-UvrC both expected incisions were observed. This means that Ct-UvrA and UvrB used in\nthis study are functional in driving proper incision by UvrC. Ct-UvrC may require different\nreaction conditions to perform the expected cuts. The assay is shown in Supplementary Fig.\n1b.\n(2) Following on, given you are using room temperatures with a thermophilic enzyme, what\nare the expectations regarding the structure of the DNA when hotter, i.e. the working temp for\nthis enzyme? I would really value a discussion of this topic, here modeling would be\nwelcome.\nC. thermocellum can grow at temperatures between 50 and 68°C (PMID: 25207268), so its\nenzymes function at this temperature. The melting temperature of the DNA duplexes used for\nUvrA structure determination in this study are 75 ºC for flu-dT: and 77.5 ºC for unmodified\nDNA. We would therefore assume that in the temperatures in which our UvrA protein\nfunctions the stretch of the DNA visualized in our structures would maintain its structural\nintegrity. We also note that our incision assays were performed at 65 ºC and the proteins\nshowed the expected activity.\nWe must admit that modeling local DNA disturbances at higher temperatures is beyond our\nexpertise. It would probably require careful molecular dynamics simulations.\n(3) The final conclusions suggest that ATP is not important for damage recognition, but it\nshould be worth noting that ATP hydrolysis is necessary for UvrB loading. What would be\nreally supportive of these suggestions would be a comparison of UvrB loading with and\nwithout ATP, or even using their own expts (pull down) showing different lesions do or do not\nrequire ATP.\nThis is a very helpful comment that prompted us to perform additional experiments which\nimprove our manuscript. We performed pull-down experiments in the presence of ATP. We\nused wt UvrB and its K44A variant which is unable to hydrolyze ATP. We reasoned that\nblocking the translocation of UvrB on DNA would help us capture the complex corresponding\nto the UvrA:UvrB:DNA:AMPPNP structure we describe in the paper. The K44A mutant\nshowed slightly more efficient loading on DNA than the wild-type UvrB. The results for this\nvariant are shown in the revised version of the manuscript.\nIn the presence of ATP we observed preferential loading of UvrB on modified versus\nunmodified DNA (new panels Fig. 7c-h). The efficiency was much lower than in the presence\nof AMPPNP, which is in agreement with published data showing stable association of UvrA\nwith DNA in the presence of non-hydrolyzable analogs of ATP (PMID: 30020831)\nIn these experiments we also tested two variants of UvrB with substitution of the wedge\nresidue (Y62A) and a residue located nearby (N270A). N270A showed loading\nindistinguishable from the wild-type UvrA (not shown). For Y62A the ATP-dependent loading\n8\nof UvrB on modified DNA was compromised and the specificity for the modification was lost\n(Fig. 7h).\n(4) Ref 24 is a mess!\nThis reference to the original scientific report on the changes to the systematics and\nnomenclature of this species was not crucial and was removed from the text.\n(5) Line 954 reference to 57 is incorrect – that study was in vivo, do the authors mean to use\nref 59 here?\nThe text has been corrected.\n(6) Line 934: An interesting observation here is that end-binding occurs with undamaged\nDNA. This suggests that UvrA would have transient interactions along the DNA looking for\ndamage, until it finds something that is more stable, such as the Flu-dT, what is the relative\npreference for damage in the middle of the DNA vs the end? This should be inherent with the\ndistribution of particles I would have thought?\nThis is an interesting suggestion. Indeed, reliable quantification of the number of particles\ncorresponding to end binding and stable interaction with flu-dT would be very informative.\nHowever, this is not straightforward. There is no well-defined DNA density in the center of\nsome cryo-EM maps, in particular those corresponding to the basal conformation. This can\nbe explained either by end binding or flexibility of the DNA in the center. These two situations\nare difficult to distinguish in our data sets. So, unfortunately, we cannot provide such\nstatistics with confidence.\nAnother interesting possibility is that the clamping of the DNA by DNA-binding domains in the\nbasal conformation is the part of the initial damage search by UvrA. We mention this\npossibility in the text:\n“An intriguing finding from our structural work is that the DNA-binding domains of UvrA clamp\nthe DNA in straight configuration but do not stably bind the bent duplex. It is possible that\nclamping of the straight duplex corresponds to the initial ATP-independent DNA binding. This\nwould be in agreement with iSCAT data that showed two modes of UvrA:DNA association:\na more transient ATPase-independent interaction and another ATP-dependent interaction that\nleads to the formation of more stable complexes (PMID: 35311869).”\nWe also want to point out that cryo-EM maps for ATP-bound conformations show continuous\nDNA, so the vast majority of particles used for these reconstructions are not in end-binding\nmode.\n(7) Line 928: I think that this conclusion needs to be supported by data. There are a lot of\ndifferences between the 2xFlu-dT in a different species and this. Ideally, you would need to\nhave the structure of two Fl-dTs in Ct. This is not an experiment I would want to see, rather a\nmodulation of the statement!\nPrompted by this comment and to better understand the differences between cryo-EM and\ncrystal structures we determined a structure of UvrA bound to a palindromic DNA with two\nflu-dT residues (not shown). This reconstruction showed a basal conformation with straight\nof the DNA in the original 3PIH crystal structure of the UvrA-DNA complex.\nIn addition, prompted by another comment, we performed 3DFlex analysis of our structures.\nThese showed malleability of UvrA:DNA complexes, in particular the DNA (new\nsupplementary Video 1) which is in agreement with the possibility that crystal packing could\naffect the conformation of the DNA.\nAs suggested we removed sentences “Another possibility is that two fluorescein\nmodifications in the self-annealing DNA that was used to determine the crystal structure of\nTm-UvrA:flu-dT(2)-DNA induce larger preexisting deformations in the DNA.” and “The ATP-\ndriven conformational change in the UvrA dimer is not required to load UvrB on the DNA.\nThis mode of the recognition was likely captured in the crystal structure of Tm-UvrA”. We\nadded sentence “Studies with other DNA modifications are needed to verify this possibility.”\n(8) Line 909, but really a concept that sits throughout the study: Given that mixed nucleotides\nare the most likely occupiers of the ATPase site (see the studies from the Hignorani group)\nhpw relevant is the saturation with a single nucleotide species?\nSeveral lines of evidence we present show that both ATPase active sites need to be\noccupied by ATP to induce the ATP-bound conformations. For example, we present\nstructures for UvrA variants in which we mutated either of the active sites of UvrA to prevent\nnucleotide binding (K39A and K647A). In both cases the proteins adopt basal conformation\nalso in the presence of AMPPNP. Also, in the presence of ADP basal conformation is\nobserved. So, among the configurations we studied only in the presence of DNA and\nAMMPNP bound at both active sites we could observe ATP-bound conformations 1 and 2.\nWe agree that other states should also be studied such as ADP/ATP mixed states. We have\nperformed preliminary pull-down experiments with two mutants of UvrA – E516A (proximal\nATPase active site) and E858A (distal site). These proteins can bind ATP but cannot\nhydrolyze ATP in respective active sites. In experiments with both mutants and UvrB K44A,\nthe DNA was pulled down efficiently, but the specificity for modified DNA was lost. So, these\nmutants mimic the effect of using AMPPNP quite closely. This suggests that ATP/ADP mixed\nstates adopt ATP-bound conformations. This also shows that both intact ATPase active sites\nneed to cooperate in ATP-dependent lesion recognition. The lack of damage specificity of\nE516A and E858A variants is in agreement with the in vivo single-molecule work of Stracy et\nal (PMID: 27562541, Fig. 6b). Overall, as explained above, we provide snapshots of a\ndynamic lesion recognition process the full understanding of which would require\ncomplementary approaches.\nWe have added the following paragraph on page 20.\n“We have characterized ATP-bound conformations 1 and 2 which are only observed when\nboth active sites are occupied by AMMPNP. Importantly, mixed ATP/ADP states were also\nobserved for UvrA (PMID: 30892613). Such states can be captured by substitutions of the\nactive sites (E516A in the proximal active site and E858A in distal active site, in Ct-UvrA\nnumbering). Substituted active sites can bind ATP but cannot hydrolyze ATP. As shown by in\nvivo single molecule studies, these UvrA variants interact tightly with DNA but lose the ability\nto discriminate the damage (PMID: 27562541). This is reminiscent of the effect of AMMPNP\nin in vitro assays, which would suggest that mixed ATP/ADP states can adopt the ATP-bound\nconformations and hydrolysis of both ATP molecules or dissociation of one ADP molecule are\nrequired for the UvrA to return to the basal conformation. Verification of this notion will require\nfurther structural studies.”\n10\n(9) I was a little surprised that UvrB loaded so strongly, such that it could resist 1M NaCl, the\ncurrent hypothesis is that UvrB needs an ATP turnover to engage the b-hairpin, so how does\nthat reconcile with the AMPPNP observations here? A wider discussion of this point would be\nreally welcome, especially since the AMPPNP data collected affects both UvrB and UvrA.\nThis is an important consideration and this was also raised by Reviewer #1.\nOne point to clarify is the definition of UvrB loading. There are two possible states. In one\nstate the β-hairpin can clamp a DNA strand away from the DNA damage. We assume this\nconfiguration arises right after UvrB is loaded on the DNA by UvrA - the state visualized by\nour UvrA:UvrB:DNA complex structure. In the structure β-hairpin clearly clamps the DNA. To\nvisualize this we added insets with close-ups of the β-hairpin and cryo-EM potential maps to\nFig. 7a, b (previously 5A, B).\nThe other state is the one in which the hairpin is locked at the site of DNA modification. Our\nUvrA:UvrB complex does not represent this state. As pointed out by the referee this requires\nATP hydrolysis. We assume that this hydrolysis is required for the UvrB to reach the lesion\nby performing ATP-dependent translocation on DNA (see PMID: 31739207) So, the UvrB\nloading according to the second definition (engagement of the β-hairpin at the modification)\nclearly requires ATP hydrolysis. We would therefore argue that our results and their\ninterpretation is in agreement with the available data, in particular with the paper of Kraithong\net al. (PMID: 33302090, Fig. R1). This information has been added to the discussion on Page\n22:\n“Our UvrA:UvrB:DNA structure was determined in the presence of AMPPNP, so the formation\nof a complex with UvrB clamping one of the DNA strands was independent of ATP hydrolysis.\nMoreover, our pull-down experiments in the presence of AMPPNP for wild-type UvrB and for\nATPase-deficient variant of UvrB (K44A) showed the formation of high salt concentration-\nresistant UvrB:DNA complex. We assume that this very stable complex involves clamping of\nthe DNA strand, as shown by our UvrA:UvrB:DNA complex structure. So, we do observe the\nATP hydrolysis-independent formation of UvrB:DNA complex. In agreement with this, several\nworks showed that in absence of ATP or for K45A mutant of UvrB (E. coli and G.\nstearothermophilus numbering), which is unable to hydrolyze ATP, UvrA:UvrB:DNA complex\nis formed (PMID: 16282327, 2671996, 33302090, 20227373).”\nWe cannot exclude the possibility that the loading of β-hairpin on the DNA is possible in our\nsystem because we use synthetic DNA duplexes with blunt ends which can serve as entry\npoints for UvrB. However, our preliminary results showed that UvrB can also be loaded on\nplasmid DNA which makes this possibility less likely.\n(10) Line 821: DO the authors agree that an alternative hypothesis to their results on the\nlysine ATPase knockout mutants could be that AMPPNP needs to be in both distal and\nproximal at the same time?\nWe hope we understand this comment correctly, but we do believe that occupancy of both\nactive sites by ATP (or potentially ADP/ATP mixed state as explained in point 8) is required\nfor the adoption of ATP-bound conformation by UvrA.\nThe sentence on page 13 reads:\n“This means that DNA binding and the presence of AMPPNP at both active sites promote the\nformation of ATP-bound conformations by UvrA (Supplementary Figure 18)”\n11\n(11) Have the authors any idea about UvrB loading with the Y62 and N270 area mutations?\nMy guess would be that UvrB couldn’t load, do they have any data (e.g. pull down) that could\nadd to that part of their (very interesting) story?\nAs described in point 3. above, we tested a variant of UvrA with substitution of the wedge\nresidue Y62 and the loading of UvrB on modified DNA was compromised. This agrees with\nthe suggestion of the Referee that the wedge is important for ATP-dependent damage\ndiscrimination. These results are now shown in Fig. 7. We also tested N270A mutant and it\nbehaved essentially as wild-type UvrA (not shown).\n(12) Is it possible that endogenous UvrA or UvrB could complex with the Ct orthologues?\nWe think this is very unlikely. The maps for the protein in most of our structures are well-\ndefined and exclude the possibility of the co-purification of endogenous Ec proteins (Fig. R2).\nFigure R2. Examples of cryo-EM potential maps for region of the Ct-UvrA and Ct-UvrA:UvrB\nstructures. Atomic models are shown in orange. Corresponding regions of Alphafold models\nof Ec proteins are superimposed (cyan).\n12\n(13) Could the authors explain why cross-linking was needed? Also, could they make it\nclearer in the results that these structures were cross-linked, rather than only mentioning it in\nthe methods.\nWe also prepared samples for Ct-UvrA without cross-linking. However, there were\nsignificantly fewer good particles per micrograph. The maps resulting from non-crosslinked\nsamples were very similar to those from cross-linked samples; however, the resolution of\nthese maps was lower (Fig. R3).\nFigure R3. Cryo-EM reconstructions of Ct-Uvr:flu-dT-DNA obtained from samples after\nchemical cross-linking (left) and without this step (right).\nThis information is now included both in the Methods and Results sections.\nReviewer #3 (Remarks to the Author):\nNucleotide excision repair (NER) is a critical pathway for the maintenance of genome stability\nin response to various types of DNA damage. A key step in this pathway is the recognition of\ndamage and subsequent recruitment of downstream effector proteins. In bacteria, DNA\ndamage is recognized by the ATPase UvrA, which initiates the NER pathway by recruiting\nUvrB. The manuscript by Nirwal et al. presents a detailed structural analysis of DNA-\nengagement by UvrA and recruitment of UvrB, giving important insights into key steps in the\nbacterial NER pathway. The manuscript is built around a series of cryogenic electron\nmicroscopy (cryo-EM) structures (14 deposited PDBs) of UvrA +/- nucleotides, +/- DNA and\n+/- UvrB that illustrate distinct UvrA/DNA conformational states. The cryo-EM work is solid\nand the high-resolution of the structures (albeit with reduced resolution in the DNA) allow the\nauthors to propose a mechanism for how DNA damage is recognized by UvrA.\nThe cryo-EM data represents a thorough, detailed analysis of UvrA interactions with DNA\nunder a range of mechanistically relevant conditions, the manuscript, figures and\naccompanying figure legends are clear and include important acknowledgement of certain\nlimitations of the data (e.g. weak DNA density in some regions of the cryo-EM maps; possible\n‘end-binding’). The data is well supported by a comprehensive ‘Materials and Methods’\nsection and supplementary materials (incl. an excellent movie summarizing the structural\n13\nstates observed in the EM data). Overall, this is an impressive piece of work and it is evident\nthat the authors have taken great care and consideration in preparing the manuscript.\nWe are very glad to hear that this Reviewer thinks highly of our work.\n1. The structures and accompanying data offer important insights into the interactions\nbetween UvrA, DNA and UvrB and how different functional states relate to nucleotide\nbinding. These structures, viewed in the context of previously published data allow the\nauthors to speculate about the mechanism by which UvrA specifically recognizes sites of\nDNA damage. However, the data presented here, as it stands, does not demonstrate specific\nbinding to (or preference for) damaged DNA, or distinct structural features that might explain\npreferential binding to damaged DNA. On the contrary, the structures and biochemical\nassays (e.g. UvrB loading) appear to show little difference between unmodified and modified\nDNA, making many of the key statements of conclusions (incl. the title) difficult to support.\nFor example, the statement “our results present a complete description of a unique\nmechanism of DNA damage detection by …” overstates what can be concluded from the\ndata presented. This is not to diminish the quality of the work, the value of the structures or\nthe merit of publication, but the conclusions should be reasonable given the data presented\nand I would recommend careful consideration of what can be concluded.\nPrompted by this comment and other comments of the Reviewers, we performed pull-down\nexperiments in the presence of ATP. They are shown in updated Fig. 7c, d, e, f. These results\nshowed that the presence of ATP promoted preferential loading of UvrB on modified versus\nunmodified DNA. We also tested two variants of UvrA with substitution of the wedge residue\nY62 and a nearby residue N270. For Y62A substitution the loading of UvrB on modified DNA\nin the presence of ATP was compromised and specificity for modified DNA was lost. This\nagrees with the importance of the wedge for ATP-dependent damage discrimination. N270A\nshowed loading indistinguishable from the wild-type UvrA (not shown).\nA similar comment about tempering of some of our statements was made by Referee 1. We\nagree that we provide snapshots of the process and additional studies will be needed to fully\ncharacterize the mechanism. We carefully went through the text and softened the language.\nWe also change the title to:\n“Structural snapshots of the mechanism of ATP-dependent DNA damage recognition by\nUvrA”\nAs mentioned above, we also added the following sentence at the end of the Discussion\nsection:\n“The damage recognition process by bacterial NER is clearly dynamic. Our structural data\nprovide snapshots of key steps this process. Its full understanding will require additional\nstudies guided by our structural data which will include time-resolved cryo-EM, single-\nmolecule biophysics and molecular dynamics simulations. Together with the structure\npresented herein they will provide a detailed comprehensive picture of damage recognition\nby bacterial NER.”\n2. The main distinguishing feature in the data between damaged and undamaged DNA\n14\nappears to be the presence of ATP-bound conformation 2, which was identified for the abasic\nand unmodified DNA but not for flu-dT DNA. Whilst noted, little comment is made on this\nobservation. Related to this, in the EM map for ATP-bound conformation 2 with unmodified\nDNA (EMD-51669), density appears to be present for the flipped base, but this is not built\ninto the current models (see attached images). This density is absent in the DNA containing\nan abasic site (albeit the sharpened map is noisier). Is it possible that recognition of certain\ntypes of DNA damage is related to an inability to form ATP-bound conformation 2 (potentially\nwith consequences for ATP hydrolysis and substrate release)?\nThis is a valid point. Our comment on page 11 was extended by an additional sentence, so\nthat the whole paragraph reads:\n“One interesting consideration is why ATP-bound conformation 1 dominated in our cryo-EM\nsample for flu-dT DNA, whereas two ATP-bound conformations were observed for abasic and\nunmodified DNA. This may be attributable to different deformability or preexisting\ndeformations in each of the DNAs we used. flu-dT can be the most flexible DNA in our set;\nwhen UvrA binds it, it readily adopts ATP-bound conformation 1. For abasic DNA, its different\ndynamic properties can drive the formation of both ATP-bound conformations 1 and 2. As\ndescribed above, cryo-EM potential maps of ATP-bound conformation 2 suggest binding of a\nflipped out base by UvrA. Perhaps this conformation is preferred on DNAs in which the\nmodification allows such binding of a flipped out base. In sum, the ability of UvrA to adopt\ndifferent ATP-bound conformations may facilitate the recognition of different DNA lesions.”\nIndeed, there is a density near base of nt 17 located at the wedge which could correspond to\na flipped-out base. We have attempted to build an atomic model there, but this resulted in\nsevere steric clashes, so we did not include this flipped out base in the final model. However,\nto note this, we added the following paragraph on page 10.\n“In the ATP-bound conformation 2 reconstruction we observed density near the ribose ring of\nnt 17 located at the center of the DNA. This density is located between the backbone of S61\nand side chain of F755 and could correspond to a flipped-out base. However, adding the\nbase to our atomic model resulted in steric clashes with the protein. Therefore, we decided to\ninsert an abasic nucleotide in the structure in this position. Further structural studies and the\ndetermination of higher-resolution structures will be required to visualize the base flipping in\nthis position.”\n3. Related to the above, lines 588-589 are not clear – is the abasic DNA used in this study a\ntarget for the NER system studied here? Does this substrate represent a genuine target for\nUvrA or a ‘control’ non-target sequence, like the unmodified DNA?\nTo better explain this we edited this part of the text to read:\n“The second DNA contained a single abasic site in the middle of the sequence. This\nmodification should have a destabilizing effect on the DNA structure and should increase its\ndeformability. UvrA binds avidly to DNA containing an abasic site as compared to non-\ndamaged DNA, so we used this modification as another model DNA recognized by UvrA. It\nneeds to be noted that UvrB is poorly loaded on abasic site DNA and thus this lesion is not\nincised efficiently (PMID: 2067017). However, bacterial NER can repair base-excision repair-\nresistant chemically heterogeneous abasic sites (PMID: 31226203, 7991608, 20232790,\n2207104, 2690930).”\n15\n4. C2 symmetry is applied in the processing of all EM structures, could the authors please\nconfirm that the processing in C1 has been investigated, and the application of symmetry\ndoes not influence the maps, in particular the DNA density?\nWe calculated most of the reconstructions also in C1 and there was no substantial difference\nbetween C2 and C1 maps (Fig. R4). This information was added to Materials and Methods\nsection.\nFigure R4. Examples of cryo-EM potential maps for DNA in representative structures,\ncalculated either with applying C2 symmetry (left) or without symmetry (right).\n5. In the materials and methods, the authors mention performing a 3DFlex analysis\ncomparing nucleotide bound and free states, but this data is currently not included in the\npaper. This would be interesting to see as a supplement, to compare the relative flexibility of\ndifferent states.\n3DFlex analysis is now included as Supplementary Video 1.\n6. In the 2-aminopurine base flipping experiments, the authors plot the % change in\nfluorescence, rather than absolute values. They suggest that the larger increase in\nfluorescence for the unmodified DNA may be because there is “no pre-existing perturbations\nof base pairing in this DNA”. Is the baseline fluorescence significantly higher in the flu-dT\n‘unmodified’ DNA is more amenable to flipping and this is important for distinguishing between\nundamaged and damaged DNA. For example, if ATP-bound state 2 cannot be formed by large\nadducts on a base, base flipping might more difficult on this substrate despite the fact that base\npairing is disrupted by the damage.\nThis comment allowed us to reinterpret the difference in 2-AP fluorescence change for\ndifferent DNAs. The absolute values of fluorescence were in fact 1.6-fold lower for the oligo\nwith 2-AP and flu-dT (oligo AP-1) than for the DNA with 2-AP only (oligo AP-2). The exact\nsource of these differences is not clear. However, in the light of this, our initial interpretation\ninvolving of preexisting deformations is not supported.\nHowever, thanks to the comment of this Referee, we realized that there is another\nexplanation. As the Referee suggests, differences in ATP-bound conformations 1 and 2 may\nbe responsible for the obtained results. AP-1 corresponds more closely to flu-dT oligo in our\nstructural studies and AP-2 to unmodified DNA. In our structures UvrA:flu-dT complex\npredominantly adopts ATP-bound conformation 1, while for the complex with unmodified DNA\nconformations 1 and 2 are observed. In each ATP-bound conformation the environment of\nthe flipped-out base is different. In ATP-bound conformation 2 it may be stabilized by tight\nprotein interactions (the unmodeled density described above). This different environment\ncould explain different change of the signal or the flipped out 2-AP for the two DNAs. We\nadded this information on page 12.\n7. Figure 4A and B could be significantly improved to better display the wedge element that\ncauses base flipping. The current figure is very hard to interpret and this key feature of the\nstructures should be clearly highlighted.\nWe have prepared a new version of these panels to show the structure of the wedge more\nclearly. We rendered the panels for Fig. 4a with Ct-UvrA:abasic-DNA:AMPPNP (ATP-bound\nconformation 1), owing to the better resolution of the corresponding map as compared to Ct-\nUvrA:flu-dT-DNA:AMPPN (ATP-bound conformation 1).\n8. In the discussion the authors mention UvrA having a ‘moderate specificity for modified\nDNA’ (Lines 919 and 942), it would help the reader to be more specific.\nWe decided to remove this sentence, because it was not clear, indeed. There are other\nsentences in the text on page 21 which explain this more clearly:\n“UvrA in the absence of ATP has moderate specificity for modified DNA (PMID:21240268).\nHowever, some studies have shown that when ATP is present, this specificity is much higher\n– UvrA’s affinity for damaged DNA is 103- to 104-fold higher than for unmodified DNA (PMID:\n1827034).”\n1) While our paper was being revised a new work was published describing structural\nstudies of UvrA:DNA and UvrA:UvrB:DNA complexes using proteins from M.\ntuberculosis (Mt) (PMID: 40210888). We added the following paragraph to the\nDiscussion to refer to this new work.\n“While our paper was being revised a new work was published describing structural studies\nof UvrA:DNA and UvrA:UvrB:DNA complexes using proteins from M. tuberculosis (Mt) (PMID:\n40210888). This paper describes three main structures: Mt-UvrA:DNA (3.2 Å resolution), Mt-\nUvrA2:UvrB:DNA (4.3 Å resolution) and Mt-UvrA2:UvrB2:DNA (3.7 Å resolution). We note\ndifferences in the DNAs used in our study and the work by Genta et al. One fluorescein\nmodification in one of the strands was used in current study and two symmetric\nmodifications, one in each strand, in the published study. Moreover, the UvrA:UvrB complex\nwas reconstituted using a DNA with two flu-dT nucleotides and ssDNA 5ʹ overhangs.\nNevertheless, Mt-UvrA:DNA and Mt-UvrA2:UvrB:DNA structures are very similar to our\nstructures of Ct-UvrA:DNA complexes in basal conformation, including the trajectory of the\nDNA.\nThe key feature observed in Mt-UvrA:DNA structure is that the two DNA-binding domains\n(termed Insertion Domains or IDs in the work by Genta et al.) clamp the DNA strands.\nThis happens in a manner nearly identical to the one observed in our basal conformation\nstructures. The density for the DNA near the DNA-binding domain was less well defined and\nwas interpreted by the authors as a DNA in which one strand has a flipped-out base\naccommodated in a pocket in one of the DBDs. They propose that this is the lesion\nrecognition state. Our data point to the key role of conformational changes of UvrA driven by\nthe ATPase cycles and the formation of the wedge element in damage recognition. This is\nfurther supported by our 2-AP fluorescence measurements implying base flipping (Figures\n4c, d, e, f). Furthermore, studies of Y62A variant in which substitution of a key wedge residue\ncompromised ATP-dependent damage discrimination (Figures 7g, h). To further establish the\nrole of DBD in damage recognition postulated by Genta et al, studies of ATP-dependent\ndamage discrimination of variants with substitutions in this domain will be required. It is\npossible that both mechanisms (base flipping by the wedge or by DBD) exist and cooperate\nin recognition of various types of DNA lesions.\nMt-UvrA2:UvrB2:DNA complex structure reported by Genta et al. and our Ct-\nUvrA:UvrB:DNA:AMPPNP complex superimpose well. Interestingly, the conformation of Mt-\nUvrA observed in Mt-UvrA2:UvrB2:DNA structure corresponds to ATP-bound conformation 1\nwe defined in this work. This implies that after UvrA:UvrB complex formation the ATP-bound\nconformation 1 may be stabilized by the presence of UvrB even after ATP is hydrolyzed.\nOverall, the published work and the results presented herein provide complementary\nstructural information for the mechanism of UvrA:UvrB complex.”\n18\n2) In addition, although the structures presented in the original manuscript were of good\noverall quality, we made an additional effort to improve their statistics, most\nimportantly the clash score, model-to-map correlation, and Q-score. In particular we\nremoved a few regions for which the cryo-EM density was not sufficiently well-\ndefined. We re-rendered all figures with updated coordinate files, and we uploaded\nthe new models and validation reports to the Zenodo repository along with the original\nmodels and validation reports. The tables with structure statistics have been updated\nand PDB validation reports for improved structures are also attached to this\nsubmission.\nNirwal S. et al., Structural snapshots of the mechanism of ATP-dependent DNA damage\nrecognition by UvrA.\nBelow we provide a point-by-point response with a description of the changes introduced in\nthe manuscript. We are happy to hear that all the concerns raised by the reviewers are satisfied\nin the revised manuscript.\nReviewer #1 (Remarks to the Author):\nThe authors have thoroughly responded to the concerns raised by the three reviews, and\nperformed a critical experiment to help mitigate a specific concern. The manuscript and overall\nstudy has been strengthened. I have no further concerns.\nWe are delighted to hear that all the concerns of the reviewer are satisfactorily addressed in\nthe revised manuscript.\nReviewer #2 (Remarks to the Author):\nIn this revised version of the manuscript the authors have performed additional experiments to\nsupport many of the statements made from the original. This greatly improves the study.\nWe are very glad to hear about the positive assessment of the revised manuscript by the\nreviewer.\nThere are only a couple of small changes required in my opinion:\n1. Check the use of AMMPMP instead of AMPPNP, the former was found a few times in the\nmanuscript.\nWe apologize for the typological error. We now correct the name of nucleotide to ‘AMPPNP’.\n2. Line 279-280 \"high affinity of UvrA for both damaged and undamaged DNA in the presence\nof non-hydrolyzable...\" is based on citations below, however none actually support this\nstatement. My suggestion is that the authors consider how to interpret their change in buried\nsurface in abother way, or remove the paragraph entirely:\nWagner, K., Moolenaar, G., van Noort, J. & Goosen, N. Single-molecule analysis reveals two\nseparate DNA-binding domains in the Escherichia coli UvrA dimer. Nucleic Acids Res 37,\n1962–850 Mazur, S. J. & Grossman, L. Dimerization of Escherichia coli UvrA and its binding\nto undamaged and ultraviolet light damaged DNA. Biochemistry 30, 4432–43 (1991).72 (2009). Van Houten, B., Gamper, H., Hearst, J. E. & Sancar, A. Analysis of sequential steps of\nnucleotide excision repair in Escherichia coli using synthetic substrates containing single\npsoralen adducts. Journal of Biological Chemistry 263, 16553–16560 (1988). Seeberg, E. &\nSteinum, A. L. Purification and properties of the uvrA protein from Escherichia coli. Proc Natl\nAcad Sci U S A 79, 988–92 (1982).\nremoved.\nReviewer #3 (Remarks to the Author):\nI would like to thank the authors for their thorough response to the reviews. I feel that all of my\nconcerns have been addressed.\nWe are very glad to address all the concerns of the reviewer in the revised manuscript.\nADDITIONAL REMARKS\nWe made the following additional changes:\nThe main manuscript file:\n1. The affiliation of Krzysztof Skowronek and Roman H. Szczepanowski has been\nmodified to “Biophysics and Bioanalytics Facility (RRID:SCR_021630), IN-MOL-CELL,\nInternational Institute of Molecular and Cell Biology, Warsaw, Poland.”\n2. The headings of the sections are changed from Upper case to lower case.\n3. The sections have been reorganized as specified.\n4. The section ‘MATERIAL AND METHODS’, has been renamed to ‘Methods’ and\n‘Declaration of interests’ to ‘Competing Interests’.\n5. Supplementary Figure has been renamed to ‘Supplementary Fig.’\n6. Supplementary Video are renamed as ‘Supplementary Movies’.\n7. In the Data availability section, the DOI hyperlinks for the PDBs, EMDB and Zenodo\nhave been added.\n8. In the ‘Methods’, information has been added about the purification of Ct-UvrC and\nTm-UvrC and the table listing the primers for cloning and mutagenesis has been cited.\n9. The IIMCB IN-MOL-CELL Infrastructure has been acknowledged.\n10. The information about the colour of Zn has been added to the legend of Figure 1.\n11. The information about ‘n’ and source data has been added to the legend of Figure 4.\n12. The information about the colour of Mg, ‘number of experiments’, ‘p values’ and source\ndata has been added to the legend of Figure 7.\n13. The description of the Supplementary files: Supplementary Information file,\nSupplementary Movies, has been removed.\n14. References have been rearranged due to the removal of a paragraph, as suggested by\nReviewer #2.\nSupplementary Information\n1. Additional panels showing the model-to-map fits for the selected helices and beta-\nsheets, DNA substrate and ligand (AMPPNP/ADP) along with the residues forming\nbinding site, for all the structural models have been added to the Supplementary Fig.\n13.\n2. The information about source data has been added to Supplementary Fig. 1.\n3. Supplementary Figure and References has been renamed to ‘Supplementary Fig. and\nSupplementary References’.\n4. The information about total number of micrographs has been added to Supplementary\nFig. 2-12.\nsubmission.\n3"}]

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