DdrC, a unique DNA repair factor from Deinococcus radiodurans, senses and stabilizes DNA breaks through a novel lesion-recognition mechanism involving protein asymmetry and dynamic structural changes.

Made with Rinto — analyse your own content free

D. radiodurans DNA damage resistance

Deinococcus radiodurans bacteria survive high doses of DNA damaging agents, including UV-C radiation, ionizing radiation, and desiccation.

DdrC binding and DNA compaction

DdrC compacts circular dsDNA through specific interactions with single-strand (ss) breaks and circularizes linear DNA by binding to double-strand (ds) breaks.

DdrC shifts DNA mobility

Incubation of DdrC with supercoiled, relaxed, or linear PX174 plasmid dsDNA results in DNA mobility shifted into the gel well at high DdrC concentrations.

Differential mobility shift by plasmid topology

DdrC binding shifts the electrophoretic mobility of DNA differently depending on the starting plasmid topology.

DdrC mechanism of compaction

DdrC induces DNA compaction by recognizing and binding directly to DNA nicks, with the degree of compaction dependent on the number of available nicks.

Direct binding to ss-breaks

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.

Compaction requires multiple nicks

A plasmid with only one nick showed no compaction, but a plasmid with three nicks was compacted to a single, discrete species.

5' phosphate independent binding

Removing terminal 5' phosphates from nicked pUC19 with Shrimp Alkaline Phosphatase has no effect on subsequent DdrC-mediated DNA compaction.

Compaction degree correlates with nicks

The degree of compaction of nicked plasmids increases with the number of available nicks.

DdrC-bound pUC19 plasmids: Circular

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

Apparent ∆Lk values

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.

Mechanism of compaction

Each DdrC unit recognizes and binds to two nicks, bridging two distal nick sites into close spatial proximity to cause plasmid compaction.

DdrC binding to supercoiled DNA

DdrC shows significant binding to supercoiled pUC19 and ΦX174 plasmid, with a progressive band shift suggesting multiple binding sites.

DdrC circularizes linear dsDNA

DdrC circularizes linear dsDNA and compacts it in the presence of ssDNA breaks, likely by binding at double-strand break sites.

Binding to linear ΦX174 plasmid

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.

Preference for blunt ds-breaks

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.

DdrC-induced circularization

The gel migration position of bound linear pUC19 matches unbound, relaxed circular pUC19, suggesting DdrC circularizes linear DNA.

Linear plasmid compaction with nicks

DdrC induces compaction of blunt-end, linear pUC19 harboring nicks, with the degree of compaction scaling with the number of available nicks.

DdrC-bound pUC19 plasmids: Linear

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

Nick-bridging model of circularization

DdrC circularizes linear DNA by bridging ds-breaks, then compacts the circularized plasmid by bridging ss-breaks.

DdrC binding sites

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.

DdrC affinity for ssDNA vs dsDNA

Previous reports indicated higher DdrC affinity for 67-mer ssDNA than dsDNA, with binding to DNA fragments at the termini.

DdrC homodimer structural domains

DdrC exists as a homodimer in solution, composed of two distinct domains, NTD and CTD, which fold and dimerize independently.

DdrC structure determination

The structure of DdrC was solved by X-ray crystallography to gain insight into the molecular mechanism of nick detection.

Two distinct structural domains

The crystal structure reveals DdrC is composed of an N-terminal domain (NTD, M1-E110) and a C-terminal domain (CTD, P111-G231).

DdrC exists as a dimer in solution

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.

NTD and CTD independent dimerization

Both the NTD and CTD domains can dimerize independently, contributing to the overall DdrC homodimer structure.

DdrC homodimer asymmetry

The full DdrC homodimer is inherently asymmetric, with its NTD and CTD C2 axes offset by 46°, a feature confirmed across different crystal forms.

DdrC nick detection mechanism basis

Dimer asymmetry and the two structurally different binding sites form the basis of DdrC's DNA nick detection mechanism.

Apo-structure hints at binding sites

The DdrC apo-structure reveals two large patches of partial positive charge, hinting at the location of two possible DNA binding sites.

Binding site conformations

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.

Unbroken DNA binding model

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.

Nicked DNA binding model

RF2NA prediction shows nicked DNA docks to the 'closed' pocket, where a single nick increases DNA flexibility allowing this binding.

Proposed dual nick detection mechanism

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.

Functional analysis of DdrC mutants

DdrC mutations disrupting DNA binding or lesion recognition match computational predictions, highlighting the essentiality of both activities for DdrC function and UV-C resistance.

Two distinct binding events

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.

Targeted mutagenesis strategy

Targeted mutagenesis was used to selectively disrupt non-lesion-specific (LA) and lesion-specific (HA) binding activities based on computational models.

Disruption of non-lesion-specific interactions

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.

Disruption of lesion-specific interactions

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.

DdrC function in UV-C resistance

DdrC's DNA binding and lesion recognition capabilities are essential for UV-C resistance in Deinococcus bacteria.

Baseline UV-C sensitivity

D. radiodurans ΔuvsE harboring an empty expression vector shows a 94% loss in UV-resistance at 200 J/m².

WT DdrC restores UV resistance

Complementary expression of WT ddrC restores UV resistance, making the strain 210% more UV resistant than the ΔuvsE baseline at 200 J/m².

Mutant DdrC fails to restore resistance

Neither NTD-mut nor CTD-mut ddrC variants can restore UV resistance to a ddrC knockout strain.

Mutant UV resistance loss

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.

Essentiality of DdrC functions

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.

Dominant-negative phenotype

NTD-mut and CTD-mut strains exhibit significantly lower UV resistance compared to the ddrC knockout strain, indicating a dominant-negative effect.

D. radiodurans UV-C survival

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

Similarities to other nick detection mechanisms

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 vs. PARP-1 vs. Rad4/XPC

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

Biological utility of DdrC in Deinococcus

DdrC's observed behavior of immobilizing ss-breaks and compacting nicked DNA is useful for Deinococcus under DNA damaging conditions.

▸ 10 Expand

APEX

DdrC DNA repair factor mechanism

DdrC, a unique DNA repair factor from Deinococcus radiodurans, senses and stabilizes DNA breaks through a novel lesion-recognition mechanism involving protein asymmetry and dynamic structural changes.

Made with Rinto — analyse your own content free

▸ 3 Expand

CONC

D. radiodurans DNA damage resistance

Deinococcus radiodurans bacteria survive high doses of DNA damaging agents, including UV-C radiation, ionizing radiation, and desiccation.

▸ 2 Expand

DETL

Factors contributing to resistance

Factors include robust antioxidant systems and highly efficient DNA repair mechanisms unique to Deinococcus species.

DETL

Antioxidant system

An atypically high intracellular concentration of Mn2+-based antioxidant species protects the proteome from oxidative damage enzymatically and non-enzymatically.

DETL

Rapid DNA repair response

Shielding of the proteome enables D. radiodurans to respond rapidly to DNA damage via extremely efficient DNA repair mechanisms.

▸ 2 Expand

CONC

Radiation-Desiccation Response (RDR)

The RDR is activated when the bacterium senses conditions leading to DNA damage, triggering upregulation of several DNA repair proteins.

DETL

Upregulated DNA repair proteins

The RDR cascade triggers the upregulation of proteins such as RecA, UvrA/B, GyrA/B, and SSB.

DETL

Deinococcus-unique genes

Five highly upregulated genes (ddrA, ddrB, ddrC, ddrD, and pprA) are unique to Deinococcus and lack identifiable sequence homologs outside this genus.

▸ 1 Expand

CONC

DdrC protein as repair component

The protein DdrC has been identified as an important component of the DNA repair machinery.

DETL

Previously known DdrC activities

DdrC is known to bind DNA in vitro, circularize, and compact DNA fragments, but its mechanism and biological relevance were poorly understood.

▸ 2 Expand

CONC

DdrC binding and DNA compaction

DdrC compacts circular dsDNA through specific interactions with single-strand (ss) breaks and circularizes linear DNA by binding to double-strand (ds) breaks.

▸ 2 Expand

FIND

DdrC shifts DNA mobility

Incubation of DdrC with supercoiled, relaxed, or linear PX174 plasmid dsDNA results in DNA mobility shifted into the gel well at high DdrC concentrations.

DETL

Intermolecular complex formation

The shift in mobility suggests the formation of a large intermolecular complex, though its biological relevance is unclear.

DETL

Smaller complex formation

At lower DdrC concentrations (<300 nM DdrC per nM DNA), DdrC forms smaller complexes that migrate into the gel.

▸ 4 Expand

FIND

Differential mobility shift by plasmid topology

DdrC binding shifts the electrophoretic mobility of DNA differently depending on the starting plasmid topology.

DETL

Linear and supercoiled PX174 shifts

Both linear and supercoiled ΦX174 are shifted to a discrete species, but the shift for supercoiled DNA is progressive, while linear DNA is sudden.

INSG

Increased mobility of relaxed PX174

Relaxed ΦX174 plasmid shows an unexpected increase in mobility upon DdrC binding, which is unusual for DNA binding proteins.

▸ 3 Expand

JUST

Reasons for increased mobility

Increased DNA mobility could be due to the complex being more negatively charged, DNA shortening by nuclease, or topological changes reducing radius of gyration.

DETL

Not due to charge

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.

DETL

Not due to nuclease activity

Proteinase K addition to a pre-formed DdrC-DNA complex restores plasmid mobility, showing it is not due to nuclease degradation.

INSG

Compacted plasmid as fast-moving species

The fast-moving species formed upon DdrC addition is in fact compacted plasmid, as previously shown by TEM.

FIND

Compaction depends on nicks

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.

▸ 5 Expand

CONC

DdrC mechanism of compaction

DdrC induces DNA compaction by recognizing and binding directly to DNA nicks, with the degree of compaction dependent on the number of available nicks.

▸ 3 Expand

FIND

Direct binding to ss-breaks

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.

DETL

Stabilization of nicked DNA

DdrC stabilizes the nicked DNA duplex, transforming a smeared, diffuse band into a sharp, discrete bound state.

STAT

DdrC binding stoichiometry

The DNA band fully shifted at a ratio of 2 DdrC monomers per nick, suggesting DdrC binds to DNA as a dimer.

INSG

Fragment length and compaction

Binding to the 22-mer duplex results in an upwards band shift because the fragment is too short to become compacted.

▸ 2 Expand

FIND

Compaction requires multiple nicks

A plasmid with only one nick showed no compaction, but a plasmid with three nicks was compacted to a single, discrete species.

INSG

Single nick binding sufficiency

A single nick is sufficient for DdrC binding but insufficient for DNA compaction, leaving the plasmid in a circular, relaxed topology.

FIND

DdrC protects DNA from digestion

DdrC protects DNA from SNM1a exonuclease digestion, but not BglI endonuclease digestion, indicating direct binding at the ss-break site.

▸ 2 Expand

FIND

5' phosphate independent binding

Removing terminal 5' phosphates from nicked pUC19 with Shrimp Alkaline Phosphatase has no effect on subsequent DdrC-mediated DNA compaction.

JUST

Mechanism independent of 5' phosphate

Nick binding and plasmid compaction by DdrC do not rely on interactions with a terminal 5' phosphate.

INSG

Mechanical sensing of lesions

DdrC may rely on a mechanical sensing mechanism, exploiting the higher conformational freedom of a DNA duplex at ss-break sites.

▸ 3 Expand

FIND

Compaction degree correlates with nicks

The degree of compaction of nicked plasmids increases with the number of available nicks.

CMPR

DdrC-bound pUC19 plasmids: Circular

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

STAT

Apparent ∆Lk values

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.

INSG

Mechanism of compaction

Each DdrC unit recognizes and binds to two nicks, bridging two distal nick sites into close spatial proximity to cause plasmid compaction.

▸ 2 Expand

FIND

DdrC binding to supercoiled DNA

DdrC shows significant binding to supercoiled pUC19 and ΦX174 plasmid, with a progressive band shift suggesting multiple binding sites.

INSG

Binding to writhe points

DdrC likely binds to writhe points of supercoiled plasmids, inducing structures similar to DdrC-compacted DNA.

JUST

Active supercoiling vs writhe induction

It is unclear whether DdrC actively supercoils DNA or simply induces a writhe point-like structure without over- or under-winding the DNA duplex.

▸ 5 Expand

CONC

DdrC circularizes linear dsDNA

DdrC circularizes linear dsDNA and compacts it in the presence of ssDNA breaks, likely by binding at double-strand break sites.

FIND

Binding to linear ΦX174 plasmid

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.

FIND

Preference for blunt ds-breaks

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.

INSG

DdrC-induced circularization

The gel migration position of bound linear pUC19 matches unbound, relaxed circular pUC19, suggesting DdrC circularizes linear DNA.

▸ 3 Expand

FIND

Linear plasmid compaction with nicks

DdrC induces compaction of blunt-end, linear pUC19 harboring nicks, with the degree of compaction scaling with the number of available nicks.

CMPR

DdrC-bound pUC19 plasmids: Linear

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

INSG

Nick-bridging model of circularization

DdrC circularizes linear DNA by bridging ds-breaks, then compacts the circularized plasmid by bridging ss-breaks.

JUST

DdrC binding sites

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.

▸ 3 Expand

FIND

DdrC affinity for ssDNA vs dsDNA

Previous reports indicated higher DdrC affinity for 67-mer ssDNA than dsDNA, with binding to DNA fragments at the termini.

JUST

Apparent preference for ssDNA

The apparent preference for ssDNA may be a symptom of selective DNA circularization.

JUST

DNA length and binding sites

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.

FIND

No preference with shorter DNA

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.

▸ 4 Expand

CONC

DdrC homodimer structural domains

DdrC exists as a homodimer in solution, composed of two distinct domains, NTD and CTD, which fold and dimerize independently.

▸ 2 Expand

DCSN

DdrC structure determination

The structure of DdrC was solved by X-ray crystallography to gain insight into the molecular mechanism of nick detection.

DETL

Initial crystallization challenges

Initial selenomethionyl (SeMet)-derivatized DdrC crystals yielded high-quality diffraction data but insufficient anomalous signal for phasing.

DCSN

Improved phasing strategy

Additional SeMet residues (L131M, L184M) were introduced at conserved positions to facilitate phasing, yielding the 7UDI crystal structure.

▸ 3 Expand

FIND

Two distinct structural domains

The crystal structure reveals DdrC is composed of an N-terminal domain (NTD, M1-E110) and a C-terminal domain (CTD, P111-G231).

DETL

Flexible linker region

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'.

FIND

DSF analysis confirms domains

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.

FIND

Structural homology analysis

NTD and CTD regions independently align to different structures in the PDB databank, further supporting their distinction as domains.

▸ 1 Expand

FIND

DdrC exists as a dimer in solution

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.

STAT

Full-length DdrC oligomeric state

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).

▸ 3 Expand

SUBC

NTD and CTD independent dimerization

Both the NTD and CTD domains can dimerize independently, contributing to the overall DdrC homodimer structure.

FIND

NTD dimerization in degraded sample

A proteolytically degraded DdrC sample yielded a crystal structure (8U1J) composed solely of residues 1–97 from the NTD, demonstrating independent NTD dimerization.

▸ 1 Expand

FIND

NTD dimerization in solution

Explicitly expressed NTD (1–98 truncation) purified and measured by SEC-MALS also dimerizes in solution.

STAT

NTD oligomeric state

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).

▸ 1 Expand

FIND

CTD dimerization in solution

Despite low stability and initial insolubility, the CTD domain (99–231) expressed with a fusion tag also dimerizes independently in solution after cleavage.

STAT

CTD oligomeric state

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).

▸ 3 Expand

CONC

DdrC homodimer asymmetry

The full DdrC homodimer is inherently asymmetric, with its NTD and CTD C2 axes offset by 46°, a feature confirmed across different crystal forms.

INSG

Asymmetry as endogenous feature

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.

▸ 1 Expand

FIND

Residues causing asymmetry

The asymmetry is attributed to five residues (120–125) within the interdomain region, where an α6 helix is deformed in one DdrC chain.

INSG

Deformed α6 helix tension

The broken α6 helix appears to be under tension, similar to a bent spring.

▸ 2 Expand

CONC

Loaded mousetrap mechanism

Static forces within the DdrC homodimer counteract each other in a loaded mousetrap mechanism, where a deformed α6 helix acts as a loaded spring.

DETL

Holding clasp network

A strong network of salt-bridges and H-bonds between the NTD and CTD on one face forms a 'holding clasp' counteracting the tension.

INSG

Potential energy storage

The mousetrap mechanism may store potential energy used by DdrC for its biological functions, triggered by specific biochemical signals like DNA strand ends.

▸ 5 Expand

CONC

DdrC nick detection mechanism basis

Dimer asymmetry and the two structurally different binding sites form the basis of DdrC's DNA nick detection mechanism.

INSG

Apo-structure hints at binding sites

The DdrC apo-structure reveals two large patches of partial positive charge, hinting at the location of two possible DNA binding sites.

DETL

Binding site conformations

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.

▸ 1 Expand

FIND

Unbroken DNA binding model

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.

INSG

Lesion scanning state

In this conformation, the α6 helices remain 'loaded', suggesting this binding mode represents a lesion scanning state where DdrC interrogates the DNA.

▸ 4 Expand

FIND

Nicked DNA binding model

RF2NA prediction shows nicked DNA docks to the 'closed' pocket, where a single nick increases DNA flexibility allowing this binding.

DETL

Duplex deformation by CTD

CTD clasp residues (Lys-170) deform the DNA duplex, disrupting base-pair contacts and forming a π-cation interaction with a DNA base.

DETL

Nick-specific NTD contacts

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.

JUST

5' phosphate not important

The terminal 5' Phosphate group on the nick is not predicted to form polar contacts with DdrC, consistent with experimental observations.

▸ 1 Expand

FIND

Symmetric binding for nicked DNA

RF2NA predicts a symmetric binding conformation for nicked DNA, with both binding sites in a 'closed' conformation and α6 helices in a relaxed state.

INSG

CTD interface disruption

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.

▸ 4 Expand

SUBC

Proposed dual nick detection mechanism

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.

DETL

Topological writhe point mimicry

Trapping two DNA nicks in a symmetric conformation places duplexes perpendicularly, topologically mimicking a supercoiling writhe point.

INSG

Compaction proportional to damage

DdrC progressively compacts circular DNA, with the degree of compaction proportional to the amount of DNA damage (more ss-breaks lead to more compaction).

INSG

Binding to supercoiled DNA deformations

DdrC likely recognizes similar duplex deformations (wrinkles, bubbles, kinks) that arise spontaneously in supercoiled DNA to alleviate torsional strain.

▸ 1 Expand

DETL

Mechanism for ds-break circularization

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.

JUST

Impact of 5' overhang

A 5' overhang disrupts the NTD's ability to properly engage the 3' end of the duplex, thereby reducing DdrC affinity to ds-breaks.

▸ 5 Expand

CONC

Functional analysis of DdrC mutants

DdrC mutations disrupting DNA binding or lesion recognition match computational predictions, highlighting the essentiality of both activities for DdrC function and UV-C resistance.

▸ 1 Expand

FIND

Two distinct binding events

INSG

HA vs LA binding modes

The HA binding mode corresponds to DdrC localized to specific DNA damage sites, while the LA binding mode corresponds to DdrC scanning non-specifically.

DCSN

Targeted mutagenesis strategy

Targeted mutagenesis was used to selectively disrupt non-lesion-specific (LA) and lesion-specific (HA) binding activities based on computational models.

▸ 1 Expand

FIND

Disruption of non-lesion-specific interactions

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.

JUST

CTD as core binding domain

This finding supports computational models predicting the CTD domain harbors 'core' DNA binding residues essential for binding any DNA ligand.

▸ 3 Expand

FIND

Disruption of lesion-specific interactions

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.

FIND

NTD-mut effects on HA 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.

JUST

R14/R81 ss-break specificity

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.

INSG

CTD-NTD cooperation in detection

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.

▸ 7 Expand

CONC

DdrC function in UV-C resistance

DdrC's DNA binding and lesion recognition capabilities are essential for UV-C resistance in Deinococcus bacteria.

FIND

Baseline UV-C sensitivity

D. radiodurans ΔuvsE harboring an empty expression vector shows a 94% loss in UV-resistance at 200 J/m².

FIND

WT DdrC restores UV resistance

Complementary expression of WT ddrC restores UV resistance, making the strain 210% more UV resistant than the ΔuvsE baseline at 200 J/m².

FIND

Mutant DdrC fails to restore resistance

Neither NTD-mut nor CTD-mut ddrC variants can restore UV resistance to a ddrC knockout strain.

STAT

Mutant UV resistance loss

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.

INSG

Essentiality of DdrC functions

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.

▸ 1 Expand

FIND

Dominant-negative phenotype

NTD-mut and CTD-mut strains exhibit significantly lower UV resistance compared to the ddrC knockout strain, indicating a dominant-negative effect.

JUST

Possible additional DdrC functions

The dominant-negative phenotype suggests DdrC has additional functions beyond DNA binding and lesion recognition, possibly recruiting other factors via protein-protein interactions.

CMPR

D. radiodurans UV-C survival

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

▸ 1 Expand

CONC

Similarities to other nick detection mechanisms

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.

CMPR

DdrC vs. PARP-1 vs. Rad4/XPC

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

▸ 5 Expand

INSG

Biological utility of DdrC in Deinococcus

DdrC's observed behavior of immobilizing ss-breaks and compacting nicked DNA is useful for Deinococcus under DNA damaging conditions.

JUST

Prevention of ds-breaks

Immobilization and compaction of ss-breaks by DdrC could prevent ss-breaks from becoming ds-breaks as they accumulate.

JUST

Prevention of end diffusion

In the event of a ds-break, DdrC bridges DNA ends to prevent end diffusion.

JUST

Genome supercoiling control

Trapping DNA lesions allows Deinococcus to control its genome's supercoiling state, even with ss- and ds-breaks.

▸ 2 Expand

FIND

DdrC as nucleoid-associated protein (NAP)

Previous TEM/AFM showed DdrC promotes plasmid compaction and circularization in vitro, and co-localizes with compact nucleoid DNA in vivo after γ-radiation.

JUST

Support for NAP claim

Our results support the claim that DdrC is a novel nucleoid-associated protein (NAP) maintaining compact nucleoid structure after extreme DNA damage.

INSG

Role as lesion-specific NAP

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.

INSG

DdrC interaction with other NAPs

Dominant-negative behavior of DdrC mutants suggests DdrC may interact with other NAPs or recruit repair factors via protein-protein interactions.