📝DdrC DNA repair factor mechanism
Deinococcus radiodurans bacteria survive high doses of DNA damaging agents, including UV-C radiation, ionizing radiation, and desiccation.
Factors include robust antioxidant systems and highly efficient DNA repair mechanisms unique to Deinococcus species.
An atypically high intracellular concentration of Mn2+-based antioxidant species protects the proteome from oxidative damage enzymatically and non-enzymatically.
Shielding of the proteome enables D. radiodurans to respond rapidly to DNA damage via extremely efficient DNA repair mechanisms.
The RDR is activated when the bacterium senses conditions leading to DNA damage, triggering upregulation of several DNA repair proteins.
The RDR cascade triggers the upregulation of proteins such as RecA, UvrA/B, GyrA/B, and SSB.
Five highly upregulated genes (ddrA, ddrB, ddrC, ddrD, and pprA) are unique to Deinococcus and lack identifiable sequence homologs outside this genus.
The protein DdrC has been identified as an important component of the DNA repair machinery.
DdrC is known to bind DNA in vitro, circularize, and compact DNA fragments, but its mechanism and biological relevance were poorly understood.
DdrC compacts circular dsDNA through specific interactions with single-strand (ss) breaks and circularizes linear DNA by binding to double-strand (ds) breaks.
Incubation of DdrC with supercoiled, relaxed, or linear PX174 plasmid dsDNA results in DNA mobility shifted into the gel well at high DdrC concentrations.
The shift in mobility suggests the formation of a large intermolecular complex, though its biological relevance is unclear.
At lower DdrC concentrations (<300 nM DdrC per nM DNA), DdrC forms smaller complexes that migrate into the gel.
DdrC binding shifts the electrophoretic mobility of DNA differently depending on the starting plasmid topology.
Both linear and supercoiled ΦX174 are shifted to a discrete species, but the shift for supercoiled DNA is progressive, while linear DNA is sudden.
Relaxed ΦX174 plasmid shows an unexpected increase in mobility upon DdrC binding, which is unusual for DNA binding proteins.
Increased DNA mobility could be due to the complex being more negatively charged, DNA shortening by nuclease, or topological changes reducing radius of gyration.
The increased mobility is not due to a more negative net charge as DdrC has a theoretical pI of 9.7, expecting a net charge of +5.
Proteinase K addition to a pre-formed DdrC-DNA complex restores plasmid mobility, showing it is not due to nuclease degradation.
The fast-moving species formed upon DdrC addition is in fact compacted plasmid, as previously shown by TEM.
Compaction of ΦX174 plasmid by DdrC is much greater when the plasmid has many randomly generated single-strand breaks compared to a single enzymatically-produced nick.
DdrC induces DNA compaction by recognizing and binding directly to DNA nicks, with the degree of compaction dependent on the number of available nicks.
DdrC recognizes and binds directly to ss-breaks, evidenced by a discrete band shift only when a nick is present in a 22-mer DNA duplex.
DdrC stabilizes the nicked DNA duplex, transforming a smeared, diffuse band into a sharp, discrete bound state.
The DNA band fully shifted at a ratio of 2 DdrC monomers per nick, suggesting DdrC binds to DNA as a dimer.
Binding to the 22-mer duplex results in an upwards band shift because the fragment is too short to become compacted.
A plasmid with only one nick showed no compaction, but a plasmid with three nicks was compacted to a single, discrete species.
A single nick is sufficient for DdrC binding but insufficient for DNA compaction, leaving the plasmid in a circular, relaxed topology.
DdrC protects DNA from SNM1a exonuclease digestion, but not BglI endonuclease digestion, indicating direct binding at the ss-break site.
Removing terminal 5' phosphates from nicked pUC19 with Shrimp Alkaline Phosphatase has no effect on subsequent DdrC-mediated DNA compaction.
Nick binding and plasmid compaction by DdrC do not rely on interactions with a terminal 5' phosphate.
DdrC may rely on a mechanical sensing mechanism, exploiting the higher conformational freedom of a DNA duplex at ss-break sites.
The degree of compaction of nicked plasmids increases with the number of available nicks.
The mobility of DdrC-compacted species appears 'quantized', matching specific pUC19 topoisomers in the marker.
| DNA sample | Unbound (-DdrC) Normalized mobility | Unbound (-DdrC) Linking number () | Bound (+DdrC) Normalized mobility | Bound (+DdrC) Linking number () |
|---|---|---|---|---|
| Topo marker | 0.000 | 0.00 | 0.000 | 0.00 |
| Topo marker | 0.219 | 1.00 | 0.262 | 1.00 |
| Topo marker | 0.499 | 2.00 | 0.538 | 2.00 |
| Topo marker | 0.765 | 3.00 | 0.800 | 3.00 |
| Topo marker | 1.000 | 4.00 | 1.000 | 4.00 |
| 0-nick | -0.063 | -0.19 | 0.091 | 0.32 |
| 0-nick | 0.157 | 0.67 | 0.245 | 0.92 |
| 0-nick | 0.452 | 1.82 | 0.538 | 2.07 |
| 0-nick | 0.702 | 2.81 | 0.769 | 2.98 |
A single nick results in an apparent ∆Lk of 0, two and three nicks both result in an apparent ∆Lk of 1, and four nicks result in a ∆Lk of 2.
Each DdrC unit recognizes and binds to two nicks, bridging two distal nick sites into close spatial proximity to cause plasmid compaction.
DdrC shows significant binding to supercoiled pUC19 and ΦX174 plasmid, with a progressive band shift suggesting multiple binding sites.
DdrC likely binds to writhe points of supercoiled plasmids, inducing structures similar to DdrC-compacted DNA.
It is unclear whether DdrC actively supercoils DNA or simply induces a writhe point-like structure without over- or under-winding the DNA duplex.
DdrC circularizes linear dsDNA and compacts it in the presence of ssDNA breaks, likely by binding at double-strand break sites.
DdrC binding to linear ΦX174 plasmid results in an upward band shift to a single, discrete position, indicating a fixed stoichiometric ratio of DdrC to DNA.
DdrC has a significantly higher affinity for blunt ds-breaks compared to overhangs, showing ~2-fold higher affinity than 2 nt overhangs and ~7-fold higher than 4 nt overhangs.
The gel migration position of bound linear pUC19 matches unbound, relaxed circular pUC19, suggesting DdrC circularizes linear DNA.
DdrC induces compaction of blunt-end, linear pUC19 harboring nicks, with the degree of compaction scaling with the number of available nicks.
The degree of compaction of linear plasmids incubated with DdrC scales with the number of available nicks, as seen in the apparent linking numbers.
| DNA sample | Unbound (-DdrC) Normalized mobility | Unbound (-DdrC) Linking number () | Bound (+DdrC) Normalized mobility | Bound (+DdrC) Linking number () |
|---|---|---|---|---|
| Topo marker | 0.000 | 0.00 | 0.000 | 0.00 |
| Topo marker | 0.193 | 1.00 | 0.164 | 1.00 |
| Topo marker | 0.496 | 2.00 | 0.474 | 2.00 |
| Topo marker | 0.773 | 3.00 | 0.759 | 3.00 |
| Topo marker | 1.000 | 4.00 | 1.000 | 4.00 |
| 0-nick | 0.210 | 0.84 | 0.009 | 0.04 |
| 1-nick | 0.210 | 0.84 | 0.198 | 0.81 |
| 2-nick | 0.176 | 0.71 | 0.284 | 1.16 |
| 3-nick | 0.244 | 0.97 | 0.336 | 1.37 |
| 4-nick | 0.261 | 1.04 | 0.491 | 2.00 |
| 4-nick | — | — | 0.647 | 2.63 |
DdrC circularizes linear DNA by bridging ds-breaks, then compacts the circularized plasmid by bridging ss-breaks.
The nick-bridging model implies each functional DdrC unit has two DNA binding sites capable of recognizing and binding either an ss-break or a ds-break.
Previous reports indicated higher DdrC affinity for 67-mer ssDNA than dsDNA, with binding to DNA fragments at the termini.
The apparent preference for ssDNA may be a symptom of selective DNA circularization.
A 67-mer ssDNA may contact both DdrC binding sites due to flexibility, while a dsDNA of the same length may only contact one due to its rigidity.
Re-testing with shorter 48-mer ssDNA and dsDNA ligands showed no preference for either, suggesting the 48-mer ssDNA is too short or rigid to contact both binding sites.
DdrC exists as a homodimer in solution, composed of two distinct domains, NTD and CTD, which fold and dimerize independently.
The structure of DdrC was solved by X-ray crystallography to gain insight into the molecular mechanism of nick detection.
Initial selenomethionyl (SeMet)-derivatized DdrC crystals yielded high-quality diffraction data but insufficient anomalous signal for phasing.
Additional SeMet residues (L131M, L184M) were introduced at conserved positions to facilitate phasing, yielding the 7UDI crystal structure.
The crystal structure reveals DdrC is composed of an N-terminal domain (NTD, M1-E110) and a C-terminal domain (CTD, P111-G231).
The first α-helix in the CTD (P111-A126) contains a short stretch of 17 residues with an alternate conformation, identified as a flexible 'linker'.
Full-length DdrC exhibits two melt peaks (40°C and 73°C), and isolated NTD/CTD constructs show single melt peaks matching these temperatures, indicating independent folding.
NTD and CTD regions independently align to different structures in the PDB databank, further supporting their distinction as domains.
SEC-MALS measurements confirmed that DdrC exists as a dimer in solution, with the common interaction interface identified by crystallography likely being the dimerization interface.
Full-length DdrC (1-231 FL) has a measured MW of 47.99 ± 4.14 kDa and an oligomeric state of 1.90 ± 0.16 (n-mer).
Both the NTD and CTD domains can dimerize independently, contributing to the overall DdrC homodimer structure.
A proteolytically degraded DdrC sample yielded a crystal structure (8U1J) composed solely of residues 1–97 from the NTD, demonstrating independent NTD dimerization.
Explicitly expressed NTD (1–98 truncation) purified and measured by SEC-MALS also dimerizes in solution.
The NTD (1-98) has a measured MW of 19.67 ± 1.15 kDa and an oligomeric state of 1.84 ± 0.11 (n-mer).
Despite low stability and initial insolubility, the CTD domain (99–231) expressed with a fusion tag also dimerizes independently in solution after cleavage.
The CTD (99-231) has a measured MW of 28.63 ± 5.06 kDa and an oligomeric state of 1.96 ± 0.35 (n-mer).
The full DdrC homodimer is inherently asymmetric, with its NTD and CTD C2 axes offset by 46°, a feature confirmed across different crystal forms.
The same asymmetric structure is seen in both crystal forms despite different protein contacts and chemical environments, indicating it's an endogenous structural feature, not a crystallographic artifact.
The asymmetry is attributed to five residues (120–125) within the interdomain region, where an α6 helix is deformed in one DdrC chain.
The broken α6 helix appears to be under tension, similar to a bent spring.
Static forces within the DdrC homodimer counteract each other in a loaded mousetrap mechanism, where a deformed α6 helix acts as a loaded spring.
A strong network of salt-bridges and H-bonds between the NTD and CTD on one face forms a 'holding clasp' counteracting the tension.
The mousetrap mechanism may store potential energy used by DdrC for its biological functions, triggered by specific biochemical signals like DNA strand ends.
Dimer asymmetry and the two structurally different binding sites form the basis of DdrC's DNA nick detection mechanism.
The DdrC apo-structure reveals two large patches of partial positive charge, hinting at the location of two possible DNA binding sites.
Despite involving the same residues, one potential binding site appears in an 'open' conformation while the other is in a 'closed' conformation due to asymmetry.
Computational docking shows dsDNA binds to the 'open' site, with protein-DNA interactions primarily mediated by the CTD clasp, which deforms DNA into the NTD.
In this conformation, the α6 helices remain 'loaded', suggesting this binding mode represents a lesion scanning state where DdrC interrogates the DNA.
RF2NA prediction shows nicked DNA docks to the 'closed' pocket, where a single nick increases DNA flexibility allowing this binding.
CTD clasp residues (Lys-170) deform the DNA duplex, disrupting base-pair contacts and forming a π-cation interaction with a DNA base.
DNA duplex deformation allows for nick-specific contacts within the NTD, with Arg-14 interacting with the 5' terminal base and Arg-81 binding 3' DNA backbone atoms.
The terminal 5' Phosphate group on the nick is not predicted to form polar contacts with DdrC, consistent with experimental observations.
RF2NA predicts a symmetric binding conformation for nicked DNA, with both binding sites in a 'closed' conformation and α6 helices in a relaxed state.
Disruption of the CTD dimer interface is necessary for both binding sites to be closed, suggesting the weak CTD dimer contacts plausibly break during nick detection.
DdrC binds unbroken DNA via its open face, scans for nicks by deforming DNA, and upon finding a ss-break, triggers a conformational change that opens the second binding site to trap a second nick.
Trapping two DNA nicks in a symmetric conformation places duplexes perpendicularly, topologically mimicking a supercoiling writhe point.
DdrC progressively compacts circular DNA, with the degree of compaction proportional to the amount of DNA damage (more ss-breaks lead to more compaction).
DdrC likely recognizes similar duplex deformations (wrinkles, bubbles, kinks) that arise spontaneously in supercoiled DNA to alleviate torsional strain.
The CTD forms sequence-independent contacts, pushing the ds-break into the NTD, which then makes end-specific contacts with the 5' and 3' terminal ends.
A 5' overhang disrupts the NTD's ability to properly engage the 3' end of the duplex, thereby reducing DdrC affinity to ds-breaks.
DdrC mutations disrupting DNA binding or lesion recognition match computational predictions, highlighting the essentiality of both activities for DdrC function and UV-C resistance.
Gel-based DdrC-DNA binding assays resolve two distinct events: high-affinity (HA) binding, lesion-dependent and fixed stoichiometric, and low-affinity (LA) binding, non-specific and variable stoichiometric.
The HA binding mode corresponds to DdrC localized to specific DNA damage sites, while the LA binding mode corresponds to DdrC scanning non-specifically.
Targeted mutagenesis was used to selectively disrupt non-lesion-specific (LA) and lesion-specific (HA) binding activities based on computational models.
CTD-mut and ΔCTD mutants, targeting conserved CTD residues (Arg-128, Arg-142, Arg-164, Lys-170), disrupted all DNA binding activity (LA and HA) across all pUC19 topologies.
This finding supports computational models predicting the CTD domain harbors 'core' DNA binding residues essential for binding any DNA ligand.
ΔNTD removal completely lost HA binding but retained detectable LA binding, indicating the NTD is responsible for lesion recognition while the CTD suffices for DNA binding.
Disrupting nick-recognition residues (Arg-14, Arg-81) in NTD (NTD-mut) completely lost HA binding for nicked pUC19 but had milder effects for linear and supercoiled pUC19.
This suggests Arg-14 and/or Arg-81 specifically bind ss-breaks over ds-breaks, supported by computational models where only Arg-14 recognizes ds-breaks.
Lesion recognition in NTD requires proper DNA duplex positioning by the CTD to make proper contact, as the NTD alone cannot stabilize the DdrC-DNA complex.
DdrC's DNA binding and lesion recognition capabilities are essential for UV-C resistance in Deinococcus bacteria.
D. radiodurans ΔuvsE harboring an empty expression vector shows a 94% loss in UV-resistance at 200 J/m².
Complementary expression of WT ddrC restores UV resistance, making the strain 210% more UV resistant than the ΔuvsE baseline at 200 J/m².
Neither NTD-mut nor CTD-mut ddrC variants can restore UV resistance to a ddrC knockout strain.
NTD-mut and CTD-mut variants exhibit 99.8% and 99.7% loss of UV resistance, respectively, at a UV dose of 200 J/m².
In vitro observations show NTD-mut lacks ss-break binding and CTD-mut is deficient in overall DNA binding, confirming both functions are essential for DdrC.
NTD-mut and CTD-mut strains exhibit significantly lower UV resistance compared to the ddrC knockout strain, indicating a dominant-negative effect.
The dominant-negative phenotype suggests DdrC has additional functions beyond DNA binding and lesion recognition, possibly recruiting other factors via protein-protein interactions.
WT ddrC significantly restores UV-C resistance compared to mutant or empty vector strains, highlighting its critical role in DNA repair.
| UV-C dose (J/m²) | ΔuvsE+ empty vector | ΔuvsEΔddrC+ empty vector | ΔuvsEΔddrC+ ddrC (WT) | ΔuvsEΔddrC+ ddrC (NTD-mut) |
|---|---|---|---|---|
| 0 | 1.00 ± 0.09 x 10^0 | 1.00 ± 0.13 × 10^0 | 1.00 ± 0.06 x 10^0 | 1.00 ± 0.12 × 10^0 |
| 28.7 | 5.19±0.48 x 10^-1 | 7.52 ± 1.35 × 10^-1 | 6.07 ± 1.12 x 10^-1 | 2.68 ± 0.44 x 10^-1 |
| 57.4 | 3.09 ± 0.70 × 10^-1 | 5.09 ± 1.02 x 10^-1 | 4.39 ± 1.22 x 10^-1 | 6.89 ± 0.85 x 10^-2 |
| 86.1 | 1.04 ± 0.17 × 10^-1 | 2.68 ± 0.46 × 10^-1 | 2.11 ± 0.39 × 10^-1 | 6.36 ± 3.47 x 10^-3 |
| 114.8 | 6.63 ± 1.08 × 10^-2 | 9.22 ± 0.97 x 10^-2 | 2.10 ± 0.38 × 10^-1 | 2.02 ± 1.42 x 10^-3 |
| 143.5 | 9.37 ± 5.01 × 10^-3 | 6.26 ± 1.51 x 10^-3 | 5.33 ± 2.48 × 10^-2 | 6.19 ± 5.59 x 10^-4 |
| 172.2 | 3.77 ± 1.00 × 10^-3 | 7.26 ± 3.27 x 10^-4 | 1.92 ± 0.24 x 10^-2 | 8.16 ± 3.52 x 10^-5 |
| 200.9 | 1.71 ± 0.32 x 10^-3 | 1.07 ± 0.75 x 10^-4 | 5.37 ± 0.86 x 10^-3 | 1.13 ± 0.53 x 10^-5 |
The proposed DdrC mechanism for nick detection shares similarities with PARP-1 and Rad4/XPC, but uniquely senses and traps two DNA lesions per unit.
DdrC mechanism is similar to PARP-1 and Rad4/XPC in utilizing conformational changes or scanning for lesions, but DdrC uniquely traps two lesions.
| Mechanism Aspect | PARP-1 (Human) | Rad4/XPC | DdrC |
|---|---|---|---|
| Lesion binding | Rapidly binds single-strand breaks | Binds DNA in scanning conformation | Senses and traps two DNA lesions per structural unit |
| Energy state / conformation | Loosely associated domains with high potential energy on un-nicked DNA | Attempts lower-energy protein-DNA conformation | Stored tension forces in dimer trigger conformational change |
| Detection process | Interrogates DNA via F1/F2 dimerization, F2 twists DNA upon ss-break | Interrogates DNA for lesions by flipping out DNA bases | Scans for nicks by deforming duplex via loaded α6 helix |
| Outcome/Consequence | Initiates 'structure collapse' for high affinity interaction, recruits repair factors | Kinetic barrier lower for damaged DNA (e.g., thymine dimer) | Circularization of linear DNA, compaction of nicked DNA |
DdrC's observed behavior of immobilizing ss-breaks and compacting nicked DNA is useful for Deinococcus under DNA damaging conditions.
Immobilization and compaction of ss-breaks by DdrC could prevent ss-breaks from becoming ds-breaks as they accumulate.
In the event of a ds-break, DdrC bridges DNA ends to prevent end diffusion.
Trapping DNA lesions allows Deinococcus to control its genome's supercoiling state, even with ss- and ds-breaks.
Previous TEM/AFM showed DdrC promotes plasmid compaction and circularization in vitro, and co-localizes with compact nucleoid DNA in vivo after γ-radiation.
Our results support the claim that DdrC is a novel nucleoid-associated protein (NAP) maintaining compact nucleoid structure after extreme DNA damage.
DdrC acts as a lesion-specific NAP, neutralizing ss-breaks and compacting the nucleoid proportionally to damage, aiding other NAPs in maintaining a compact nucleoid.
Dominant-negative behavior of DdrC mutants suggests DdrC may interact with other NAPs or recruit repair factors via protein-protein interactions.
Deinococcus radiodurans bacteria survive high doses of DNA damaging agents, including UV-C radiation, ionizing radiation, and desiccation.
Factors include robust antioxidant systems and highly efficient DNA repair mechanisms unique to Deinococcus species.
An atypically high intracellular concentration of Mn2+-based antioxidant species protects the proteome from oxidative damage enzymatically and non-enzymatically.
Shielding of the proteome enables D. radiodurans to respond rapidly to DNA damage via extremely efficient DNA repair mechanisms.
The RDR is activated when the bacterium senses conditions leading to DNA damage, triggering upregulation of several DNA repair proteins.
The RDR cascade triggers the upregulation of proteins such as RecA, UvrA/B, GyrA/B, and SSB.
Five highly upregulated genes (ddrA, ddrB, ddrC, ddrD, and pprA) are unique to Deinococcus and lack identifiable sequence homologs outside this genus.
The protein DdrC has been identified as an important component of the DNA repair machinery.
DdrC is known to bind DNA in vitro, circularize, and compact DNA fragments, but its mechanism and biological relevance were poorly understood.
DdrC compacts circular dsDNA through specific interactions with single-strand (ss) breaks and circularizes linear DNA by binding to double-strand (ds) breaks.
Incubation of DdrC with supercoiled, relaxed, or linear PX174 plasmid dsDNA results in DNA mobility shifted into the gel well at high DdrC concentrations.
The shift in mobility suggests the formation of a large intermolecular complex, though its biological relevance is unclear.
At lower DdrC concentrations (<300 nM DdrC per nM DNA), DdrC forms smaller complexes that migrate into the gel.
DdrC binding shifts the electrophoretic mobility of DNA differently depending on the starting plasmid topology.
Both linear and supercoiled ΦX174 are shifted to a discrete species, but the shift for supercoiled DNA is progressive, while linear DNA is sudden.
Relaxed ΦX174 plasmid shows an unexpected increase in mobility upon DdrC binding, which is unusual for DNA binding proteins.
Increased DNA mobility could be due to the complex being more negatively charged, DNA shortening by nuclease, or topological changes reducing radius of gyration.
The increased mobility is not due to a more negative net charge as DdrC has a theoretical pI of 9.7, expecting a net charge of +5.
Proteinase K addition to a pre-formed DdrC-DNA complex restores plasmid mobility, showing it is not due to nuclease degradation.
The fast-moving species formed upon DdrC addition is in fact compacted plasmid, as previously shown by TEM.
Compaction of ΦX174 plasmid by DdrC is much greater when the plasmid has many randomly generated single-strand breaks compared to a single enzymatically-produced nick.
DdrC induces DNA compaction by recognizing and binding directly to DNA nicks, with the degree of compaction dependent on the number of available nicks.
DdrC recognizes and binds directly to ss-breaks, evidenced by a discrete band shift only when a nick is present in a 22-mer DNA duplex.
DdrC stabilizes the nicked DNA duplex, transforming a smeared, diffuse band into a sharp, discrete bound state.
The DNA band fully shifted at a ratio of 2 DdrC monomers per nick, suggesting DdrC binds to DNA as a dimer.
Binding to the 22-mer duplex results in an upwards band shift because the fragment is too short to become compacted.
A plasmid with only one nick showed no compaction, but a plasmid with three nicks was compacted to a single, discrete species.
A single nick is sufficient for DdrC binding but insufficient for DNA compaction, leaving the plasmid in a circular, relaxed topology.
DdrC protects DNA from SNM1a exonuclease digestion, but not BglI endonuclease digestion, indicating direct binding at the ss-break site.
Removing terminal 5' phosphates from nicked pUC19 with Shrimp Alkaline Phosphatase has no effect on subsequent DdrC-mediated DNA compaction.
Nick binding and plasmid compaction by DdrC do not rely on interactions with a terminal 5' phosphate.
DdrC may rely on a mechanical sensing mechanism, exploiting the higher conformational freedom of a DNA duplex at ss-break sites.
The degree of compaction of nicked plasmids increases with the number of available nicks.
The mobility of DdrC-compacted species appears 'quantized', matching specific pUC19 topoisomers in the marker.
| DNA sample | Unbound (-DdrC) Normalized mobility | Unbound (-DdrC) Linking number () | Bound (+DdrC) Normalized mobility | Bound (+DdrC) Linking number () |
|---|---|---|---|---|
| Topo marker | 0.000 | 0.00 | 0.000 | 0.00 |
| Topo marker | 0.219 | 1.00 | 0.262 | 1.00 |
| Topo marker | 0.499 | 2.00 | 0.538 | 2.00 |
| Topo marker | 0.765 | 3.00 | 0.800 | 3.00 |
| Topo marker | 1.000 | 4.00 | 1.000 | 4.00 |
| 0-nick | -0.063 | -0.19 | 0.091 | 0.32 |
| 0-nick | 0.157 | 0.67 | 0.245 | 0.92 |
| 0-nick | 0.452 | 1.82 | 0.538 | 2.07 |
| 0-nick | 0.702 | 2.81 | 0.769 | 2.98 |
A single nick results in an apparent ∆Lk of 0, two and three nicks both result in an apparent ∆Lk of 1, and four nicks result in a ∆Lk of 2.
Each DdrC unit recognizes and binds to two nicks, bridging two distal nick sites into close spatial proximity to cause plasmid compaction.
DdrC shows significant binding to supercoiled pUC19 and ΦX174 plasmid, with a progressive band shift suggesting multiple binding sites.
DdrC likely binds to writhe points of supercoiled plasmids, inducing structures similar to DdrC-compacted DNA.
It is unclear whether DdrC actively supercoils DNA or simply induces a writhe point-like structure without over- or under-winding the DNA duplex.
DdrC circularizes linear dsDNA and compacts it in the presence of ssDNA breaks, likely by binding at double-strand break sites.
DdrC binding to linear ΦX174 plasmid results in an upward band shift to a single, discrete position, indicating a fixed stoichiometric ratio of DdrC to DNA.
DdrC has a significantly higher affinity for blunt ds-breaks compared to overhangs, showing ~2-fold higher affinity than 2 nt overhangs and ~7-fold higher than 4 nt overhangs.
The gel migration position of bound linear pUC19 matches unbound, relaxed circular pUC19, suggesting DdrC circularizes linear DNA.
DdrC induces compaction of blunt-end, linear pUC19 harboring nicks, with the degree of compaction scaling with the number of available nicks.
The degree of compaction of linear plasmids incubated with DdrC scales with the number of available nicks, as seen in the apparent linking numbers.
| DNA sample | Unbound (-DdrC) Normalized mobility | Unbound (-DdrC) Linking number () | Bound (+DdrC) Normalized mobility | Bound (+DdrC) Linking number () |
|---|---|---|---|---|
| Topo marker | 0.000 | 0.00 | 0.000 | 0.00 |
| Topo marker | 0.193 | 1.00 | 0.164 | 1.00 |
| Topo marker | 0.496 | 2.00 | 0.474 | 2.00 |
| Topo marker | 0.773 | 3.00 | 0.759 | 3.00 |
| Topo marker | 1.000 | 4.00 | 1.000 | 4.00 |
| 0-nick | 0.210 | 0.84 | 0.009 | 0.04 |
| 1-nick | 0.210 | 0.84 | 0.198 | 0.81 |
| 2-nick | 0.176 | 0.71 | 0.284 | 1.16 |
| 3-nick | 0.244 | 0.97 | 0.336 | 1.37 |
| 4-nick | 0.261 | 1.04 | 0.491 | 2.00 |
| 4-nick | — | — | 0.647 | 2.63 |
DdrC circularizes linear DNA by bridging ds-breaks, then compacts the circularized plasmid by bridging ss-breaks.
The nick-bridging model implies each functional DdrC unit has two DNA binding sites capable of recognizing and binding either an ss-break or a ds-break.
Previous reports indicated higher DdrC affinity for 67-mer ssDNA than dsDNA, with binding to DNA fragments at the termini.
The apparent preference for ssDNA may be a symptom of selective DNA circularization.
A 67-mer ssDNA may contact both DdrC binding sites due to flexibility, while a dsDNA of the same length may only contact one due to its rigidity.
Re-testing with shorter 48-mer ssDNA and dsDNA ligands showed no preference for either, suggesting the 48-mer ssDNA is too short or rigid to contact both binding sites.
DdrC exists as a homodimer in solution, composed of two distinct domains, NTD and CTD, which fold and dimerize independently.
The structure of DdrC was solved by X-ray crystallography to gain insight into the molecular mechanism of nick detection.
Initial selenomethionyl (SeMet)-derivatized DdrC crystals yielded high-quality diffraction data but insufficient anomalous signal for phasing.
Additional SeMet residues (L131M, L184M) were introduced at conserved positions to facilitate phasing, yielding the 7UDI crystal structure.
The crystal structure reveals DdrC is composed of an N-terminal domain (NTD, M1-E110) and a C-terminal domain (CTD, P111-G231).
The first α-helix in the CTD (P111-A126) contains a short stretch of 17 residues with an alternate conformation, identified as a flexible 'linker'.
Full-length DdrC exhibits two melt peaks (40°C and 73°C), and isolated NTD/CTD constructs show single melt peaks matching these temperatures, indicating independent folding.
NTD and CTD regions independently align to different structures in the PDB databank, further supporting their distinction as domains.
SEC-MALS measurements confirmed that DdrC exists as a dimer in solution, with the common interaction interface identified by crystallography likely being the dimerization interface.
Full-length DdrC (1-231 FL) has a measured MW of 47.99 ± 4.14 kDa and an oligomeric state of 1.90 ± 0.16 (n-mer).
Both the NTD and CTD domains can dimerize independently, contributing to the overall DdrC homodimer structure.
A proteolytically degraded DdrC sample yielded a crystal structure (8U1J) composed solely of residues 1–97 from the NTD, demonstrating independent NTD dimerization.
Explicitly expressed NTD (1–98 truncation) purified and measured by SEC-MALS also dimerizes in solution.
The NTD (1-98) has a measured MW of 19.67 ± 1.15 kDa and an oligomeric state of 1.84 ± 0.11 (n-mer).
Despite low stability and initial insolubility, the CTD domain (99–231) expressed with a fusion tag also dimerizes independently in solution after cleavage.
The CTD (99-231) has a measured MW of 28.63 ± 5.06 kDa and an oligomeric state of 1.96 ± 0.35 (n-mer).
The full DdrC homodimer is inherently asymmetric, with its NTD and CTD C2 axes offset by 46°, a feature confirmed across different crystal forms.
The same asymmetric structure is seen in both crystal forms despite different protein contacts and chemical environments, indicating it's an endogenous structural feature, not a crystallographic artifact.
The asymmetry is attributed to five residues (120–125) within the interdomain region, where an α6 helix is deformed in one DdrC chain.
The broken α6 helix appears to be under tension, similar to a bent spring.
Static forces within the DdrC homodimer counteract each other in a loaded mousetrap mechanism, where a deformed α6 helix acts as a loaded spring.
A strong network of salt-bridges and H-bonds between the NTD and CTD on one face forms a 'holding clasp' counteracting the tension.
The mousetrap mechanism may store potential energy used by DdrC for its biological functions, triggered by specific biochemical signals like DNA strand ends.
Dimer asymmetry and the two structurally different binding sites form the basis of DdrC's DNA nick detection mechanism.
The DdrC apo-structure reveals two large patches of partial positive charge, hinting at the location of two possible DNA binding sites.
Despite involving the same residues, one potential binding site appears in an 'open' conformation while the other is in a 'closed' conformation due to asymmetry.
Computational docking shows dsDNA binds to the 'open' site, with protein-DNA interactions primarily mediated by the CTD clasp, which deforms DNA into the NTD.
In this conformation, the α6 helices remain 'loaded', suggesting this binding mode represents a lesion scanning state where DdrC interrogates the DNA.
RF2NA prediction shows nicked DNA docks to the 'closed' pocket, where a single nick increases DNA flexibility allowing this binding.
CTD clasp residues (Lys-170) deform the DNA duplex, disrupting base-pair contacts and forming a π-cation interaction with a DNA base.
DNA duplex deformation allows for nick-specific contacts within the NTD, with Arg-14 interacting with the 5' terminal base and Arg-81 binding 3' DNA backbone atoms.
The terminal 5' Phosphate group on the nick is not predicted to form polar contacts with DdrC, consistent with experimental observations.
RF2NA predicts a symmetric binding conformation for nicked DNA, with both binding sites in a 'closed' conformation and α6 helices in a relaxed state.
Disruption of the CTD dimer interface is necessary for both binding sites to be closed, suggesting the weak CTD dimer contacts plausibly break during nick detection.
DdrC binds unbroken DNA via its open face, scans for nicks by deforming DNA, and upon finding a ss-break, triggers a conformational change that opens the second binding site to trap a second nick.
Trapping two DNA nicks in a symmetric conformation places duplexes perpendicularly, topologically mimicking a supercoiling writhe point.
DdrC progressively compacts circular DNA, with the degree of compaction proportional to the amount of DNA damage (more ss-breaks lead to more compaction).
DdrC likely recognizes similar duplex deformations (wrinkles, bubbles, kinks) that arise spontaneously in supercoiled DNA to alleviate torsional strain.
The CTD forms sequence-independent contacts, pushing the ds-break into the NTD, which then makes end-specific contacts with the 5' and 3' terminal ends.
A 5' overhang disrupts the NTD's ability to properly engage the 3' end of the duplex, thereby reducing DdrC affinity to ds-breaks.
DdrC mutations disrupting DNA binding or lesion recognition match computational predictions, highlighting the essentiality of both activities for DdrC function and UV-C resistance.
Gel-based DdrC-DNA binding assays resolve two distinct events: high-affinity (HA) binding, lesion-dependent and fixed stoichiometric, and low-affinity (LA) binding, non-specific and variable stoichiometric.
The HA binding mode corresponds to DdrC localized to specific DNA damage sites, while the LA binding mode corresponds to DdrC scanning non-specifically.
Targeted mutagenesis was used to selectively disrupt non-lesion-specific (LA) and lesion-specific (HA) binding activities based on computational models.
CTD-mut and ΔCTD mutants, targeting conserved CTD residues (Arg-128, Arg-142, Arg-164, Lys-170), disrupted all DNA binding activity (LA and HA) across all pUC19 topologies.
This finding supports computational models predicting the CTD domain harbors 'core' DNA binding residues essential for binding any DNA ligand.
ΔNTD removal completely lost HA binding but retained detectable LA binding, indicating the NTD is responsible for lesion recognition while the CTD suffices for DNA binding.
Disrupting nick-recognition residues (Arg-14, Arg-81) in NTD (NTD-mut) completely lost HA binding for nicked pUC19 but had milder effects for linear and supercoiled pUC19.
This suggests Arg-14 and/or Arg-81 specifically bind ss-breaks over ds-breaks, supported by computational models where only Arg-14 recognizes ds-breaks.
Lesion recognition in NTD requires proper DNA duplex positioning by the CTD to make proper contact, as the NTD alone cannot stabilize the DdrC-DNA complex.
DdrC's DNA binding and lesion recognition capabilities are essential for UV-C resistance in Deinococcus bacteria.
D. radiodurans ΔuvsE harboring an empty expression vector shows a 94% loss in UV-resistance at 200 J/m².
Complementary expression of WT ddrC restores UV resistance, making the strain 210% more UV resistant than the ΔuvsE baseline at 200 J/m².
Neither NTD-mut nor CTD-mut ddrC variants can restore UV resistance to a ddrC knockout strain.
NTD-mut and CTD-mut variants exhibit 99.8% and 99.7% loss of UV resistance, respectively, at a UV dose of 200 J/m².
In vitro observations show NTD-mut lacks ss-break binding and CTD-mut is deficient in overall DNA binding, confirming both functions are essential for DdrC.
NTD-mut and CTD-mut strains exhibit significantly lower UV resistance compared to the ddrC knockout strain, indicating a dominant-negative effect.
The dominant-negative phenotype suggests DdrC has additional functions beyond DNA binding and lesion recognition, possibly recruiting other factors via protein-protein interactions.
WT ddrC significantly restores UV-C resistance compared to mutant or empty vector strains, highlighting its critical role in DNA repair.
| UV-C dose (J/m²) | ΔuvsE+ empty vector | ΔuvsEΔddrC+ empty vector | ΔuvsEΔddrC+ ddrC (WT) | ΔuvsEΔddrC+ ddrC (NTD-mut) |
|---|---|---|---|---|
| 0 | 1.00 ± 0.09 x 10^0 | 1.00 ± 0.13 × 10^0 | 1.00 ± 0.06 x 10^0 | 1.00 ± 0.12 × 10^0 |
| 28.7 | 5.19±0.48 x 10^-1 | 7.52 ± 1.35 × 10^-1 | 6.07 ± 1.12 x 10^-1 | 2.68 ± 0.44 x 10^-1 |
| 57.4 | 3.09 ± 0.70 × 10^-1 | 5.09 ± 1.02 x 10^-1 | 4.39 ± 1.22 x 10^-1 | 6.89 ± 0.85 x 10^-2 |
| 86.1 | 1.04 ± 0.17 × 10^-1 | 2.68 ± 0.46 × 10^-1 | 2.11 ± 0.39 × 10^-1 | 6.36 ± 3.47 x 10^-3 |
| 114.8 | 6.63 ± 1.08 × 10^-2 | 9.22 ± 0.97 x 10^-2 | 2.10 ± 0.38 × 10^-1 | 2.02 ± 1.42 x 10^-3 |
| 143.5 | 9.37 ± 5.01 × 10^-3 | 6.26 ± 1.51 x 10^-3 | 5.33 ± 2.48 × 10^-2 | 6.19 ± 5.59 x 10^-4 |
| 172.2 | 3.77 ± 1.00 × 10^-3 | 7.26 ± 3.27 x 10^-4 | 1.92 ± 0.24 x 10^-2 | 8.16 ± 3.52 x 10^-5 |
| 200.9 | 1.71 ± 0.32 x 10^-3 | 1.07 ± 0.75 x 10^-4 | 5.37 ± 0.86 x 10^-3 | 1.13 ± 0.53 x 10^-5 |
The proposed DdrC mechanism for nick detection shares similarities with PARP-1 and Rad4/XPC, but uniquely senses and traps two DNA lesions per unit.
DdrC mechanism is similar to PARP-1 and Rad4/XPC in utilizing conformational changes or scanning for lesions, but DdrC uniquely traps two lesions.
| Mechanism Aspect | PARP-1 (Human) | Rad4/XPC | DdrC |
|---|---|---|---|
| Lesion binding | Rapidly binds single-strand breaks | Binds DNA in scanning conformation | Senses and traps two DNA lesions per structural unit |
| Energy state / conformation | Loosely associated domains with high potential energy on un-nicked DNA | Attempts lower-energy protein-DNA conformation | Stored tension forces in dimer trigger conformational change |
| Detection process | Interrogates DNA via F1/F2 dimerization, F2 twists DNA upon ss-break | Interrogates DNA for lesions by flipping out DNA bases | Scans for nicks by deforming duplex via loaded α6 helix |
| Outcome/Consequence | Initiates 'structure collapse' for high affinity interaction, recruits repair factors | Kinetic barrier lower for damaged DNA (e.g., thymine dimer) | Circularization of linear DNA, compaction of nicked DNA |
DdrC's observed behavior of immobilizing ss-breaks and compacting nicked DNA is useful for Deinococcus under DNA damaging conditions.
Immobilization and compaction of ss-breaks by DdrC could prevent ss-breaks from becoming ds-breaks as they accumulate.
In the event of a ds-break, DdrC bridges DNA ends to prevent end diffusion.
Trapping DNA lesions allows Deinococcus to control its genome's supercoiling state, even with ss- and ds-breaks.
Previous TEM/AFM showed DdrC promotes plasmid compaction and circularization in vitro, and co-localizes with compact nucleoid DNA in vivo after γ-radiation.
Our results support the claim that DdrC is a novel nucleoid-associated protein (NAP) maintaining compact nucleoid structure after extreme DNA damage.
DdrC acts as a lesion-specific NAP, neutralizing ss-breaks and compacting the nucleoid proportionally to damage, aiding other NAPs in maintaining a compact nucleoid.
Dominant-negative behavior of DdrC mutants suggests DdrC may interact with other NAPs or recruit repair factors via protein-protein interactions.