Recombinant Photobacterium profundum UvrABC system protein C (uvrC), partial

What is the taxonomic classification of Photobacterium profundum and how does this context inform UvrC research?

Photobacterium profundum is a marine Gammaproteobacterium belonging to the family Vibrionaceae and genus Photobacterium. Its detailed taxonomic classification includes:

P. profundum is particularly notable as a deep-sea bacterium with multiple strains adapted to different pressure and temperature conditions. For example, strain SS9 demonstrates optimal growth at 15°C and 28 MPa, making it both a psychrophile and piezophile, while strain 3TCK from San Diego Bay grows optimally at 9°C and 0.1 MPa . The ability of P. profundum to thrive in extreme conditions suggests its DNA repair systems, including UvrC, may have unique adaptations, making it an excellent model for comparative studies of DNA repair mechanisms across pressure and temperature gradients.

How does the UvrABC system function in bacterial nucleotide excision repair?

The UvrABC excinuclease system represents a critical bacterial DNA repair pathway responsible for removing a wide range of bulky DNA adducts. The process follows a well-defined sequence:

• Damage recognition: The UvrA₂B₂ heterotetramer binds to damaged DNA regions

• Complex reconfiguration: UvrA₂ dissociates from UvrB₂, allowing the latter to verify damage

• UvrC recruitment: UvrB recruits UvrC to form the pre-incision complex

• Dual incision: UvrC performs incisions on both sides of the DNA lesion, releasing a 12-13 nucleotide fragment containing the damage

• Post-incision processing: UvrD helicase and DNA polymerase I remove the oligonucleotide and fill the gap

This multi-step, ATP-dependent process removes diverse DNA lesions including cyclobutane pyrimidine dimers, alkylation adducts, and cisplatin interstrand cross-links . Understanding the UvrC component of P. profundum specifically requires consideration of how this system may be adapted to function under the high-pressure, low-temperature conditions of the deep sea environment.

What are the key structural domains of UvrC and how do they contribute to its endonuclease activity?

UvrC possesses a complex multi-domain architecture responsible for its dual incision capability:

Recent research combining structure prediction algorithms and crystallographic data has revealed that UvrC maintains a "closed" inactive state that must undergo a major conformational rearrangement to adopt an "open" active state capable of performing the dual incision reaction . The central inactive RNase H domain serves as a platform for surrounding domains, acting as a structural scaffold rather than having catalytic activity. The two HhH motifs in UvrC's C-terminal region form one functional unit involved in DNA binding, contributing to both the 3′ and 5′ incision activities .

How do the helix-hairpin-helix (HhH) motifs in UvrC influence DNA binding and repair efficiency?

The HhH motifs in UvrC play a critical role in its function:

• Structural arrangement: UvrC contains two HhH motifs that fold together to form a functional (HhH)₂ domain

• DNA binding functionality: This domain enables non-specific DNA binding, helping position UvrC correctly on damaged DNA

• Damage-dependent contribution: The importance of HhH motifs varies depending on:

  • Sequence context of the damage

  • Nature of the DNA lesion

  • Position relative to the damage site

Experimental evidence demonstrates that mutations in the HhH motifs can affect both 3′ and 5′ incision activities . The C-terminal region containing these motifs contributes significantly to the flexibility of the UvrABC system, allowing it to process various substrates with different efficiencies. This adaptability is essential for addressing the diverse range of DNA lesions encountered in bacterial environments.

What methods are most effective for expressing and purifying recombinant P. profundum UvrC?

For optimal expression and purification of recombinant P. profundum UvrC, researchers should consider:

Expression system optimization:

• Vector selection:

  • For full-length UvrC: pBL12-based vectors have been successfully used for UvrC mutants

  • For partial or domain-specific constructs: pMUT100 for insertional inactivation

• Host selection:

  • E. coli strains like S17-1λpir for conjugation experiments

  • Consider low-temperature induction methods to maintain proper folding of psychrophilic proteins

• Growth conditions:

  • Temperature: 15°C optimal for P. profundum protein expression

  • Media: Modified 2216 marine broth (28 g/liter)

  • Antibiotics: Kanamycin (200 μg/ml), chloramphenicol (15 μg/ml), or rifampin (100 μg/ml) as required

Purification approaches:

• Affinity chromatography with His-tag or other fusion tags

• Size-exclusion chromatography (SEC) for complex analysis

• Surface plasmon resonance (SPR) for interaction studies

When working with partial UvrC constructs, careful consideration of domain boundaries is essential to preserve functional integrity. For P. profundum specifically, pressure adaptation might necessitate modifications to standard protocols to ensure proper folding of the recombinant protein.

What techniques can be used to evaluate UvrC-DNA interactions and incision activities?

Several complementary approaches can be employed to study UvrC-DNA interactions:

For binding studies:

• Electrophoretic mobility shift assays (EMSA) to assess DNA binding

• Surface plasmon resonance (SPR) for quantitative binding kinetics

• Fluorescence anisotropy with fluorescently labeled DNA substrates

For enzymatic activity:

• In vitro incision assays using:

  • Substrates with defined lesions (e.g., cyclobutane pyrimidine dimers, bulky adducts)

  • 5'-labeled oligonucleotides for tracking incision products

  • Denaturing polyacrylamide gel electrophoresis for product analysis

For complex formation:

• Size-exclusion chromatography (SEC) to detect UvrA-UvrC or UvrB-UvrC complexes

• Yeast two-hybrid (Y2H) assays for protein-protein interactions in vivo

• Co-immunoprecipitation to validate interactions in cellular contexts

For P. profundum specifically, researchers should consider how pressure and temperature conditions might influence these interactions. Specialized high-pressure equipment, such as pressure vessels for bacterial cultivation , may be needed to study the protein under native-like conditions.

How does the direct interaction between UvrA and UvrC in P. profundum compare to the Mycobacterium tuberculosis model?

Recent discoveries challenge the traditional model of UvrABC function, particularly regarding direct UvrA-UvrC interactions:

M. tuberculosis UvrA-UvrC interaction:

• MtUvrA binds to MtUvrC with submicromolar affinity

• This interaction occurs independently of UvrB and DNA

• The affinity between MtUvrA and MtUvrC is comparable to that between MtUvrA and MtUvrB

Research approach for P. profundum comparison:

• Affinity measurements: Use purified P. profundum UvrA and UvrC in SPR or isothermal titration calorimetry

• Complex characterization: Apply SEC to detect complex formation under varying pressure conditions

• In vivo validation: Employ bacterial two-hybrid systems adapted for pressure studies

This comparison would provide valuable insights into how the UvrABC pathway might be modified in pressure-adapted bacteria. The direct UvrA-UvrC interaction represents a significant revision to our understanding of bacterial NER, suggesting a potential pre-recruitment mechanism that could be particularly advantageous in extreme environments where rapid DNA repair is essential for survival.

What role does UvrC play in suppressing illegitimate recombination in P. profundum, and how might this function be affected by high-pressure environments?

UvrC's role extends beyond direct DNA repair to genome stability maintenance:

Known UvrABC system functions in recombination:

• UvrA and UvrB suppress illegitimate recombination

• The uvrA uvrB double mutation increases illegitimate recombination ~30-fold relative to wild-type

• UvrA appears to function in the same pathway as RecQ helicase in suppressing illegitimate recombination

Research questions for P. profundum context:

• Does high pressure influence the rate of illegitimate recombination?

• Is UvrC's role in suppressing recombination enhanced or diminished under pressure?

• How do pressure-specific mutations in UvrC affect genome stability?

Experimental approach:

• Generate uvrC mutants in P. profundum

• Measure recombination rates using reporter systems under varying pressure conditions

• Compare recombination frequencies between wild-type and mutant strains

• Analyze genomic stability after exposure to DNA-damaging agents at different pressures

This research would elucidate how DNA repair and recombination systems are balanced in extreme environments, potentially revealing unique adaptations in P. profundum that prevent genomic instability under high-pressure stress.

How does the amino acid composition of P. profundum UvrC compare to mesophilic bacteria, and what adaptations might enable its function under high pressure?

Piezophilic (pressure-adapted) proteins typically display specific adaptations:

Expected pressure-adaptive features in P. profundum UvrC:

Research methodology:

• Comparative sequence analysis between P. profundum UvrC and mesophilic homologs

• Homology modeling incorporating pressure-specific considerations

• Site-directed mutagenesis to test the role of putative pressure-adaptive residues

• Functional assays under varying pressure conditions

Analysis of P. profundum UvrC's amino acid composition would provide insights into molecular adaptations that enable DNA repair enzymes to function efficiently in deep-sea environments. Such adaptations might be particularly evident in regions responsible for conformational changes, as the "closed" to "open" transition described for UvrC would be directly affected by pressure.

How does the UvrC-dependent repair efficiency of different DNA lesions vary under high-pressure conditions in P. profundum?

UvrABC repair efficiency varies significantly depending on lesion type:

Factors affecting repair efficiency:

• Rate of UvrA binding to damaged DNA

• Rates of UvrB-DNA and UvrBC-DNA complex formation

• Sterical hindrance affecting 3' incision by UvrC

• Stability of the UvrB-DNA complex (inverse correlation with incision efficiency)

Pressure-specific research questions:

• Does high pressure alter substrate preference of the UvrABC system?

• Are certain lesions more efficiently repaired under pressure?

• Does pressure affect the conformational changes required for UvrC activation?

Experimental design:

• Prepare DNA substrates with various lesions (e.g., cyclobutane pyrimidine dimers, cisplatin adducts)

• Perform in vitro incision assays under varying pressure conditions

• Quantify repair efficiency using gel electrophoresis and fluorescence detection

• Compare kinetic parameters derived from pressure-dependent rate measurements

This research would reveal how environmental pressure influences DNA damage recognition and repair, potentially identifying unique adaptations in P. profundum UvrC that optimize repair efficiency under deep-sea conditions.

What strategies can be employed to study the "closed" to "open" conformational transition of P. profundum UvrC?

The conformational change from "closed" (inactive) to "open" (active) state is crucial for UvrC function:

Techniques for studying conformational changes:

Pressure-specific considerations:

• Design pressure-resistant fluorophores for FRET studies

• Develop high-pressure sample holders for structural studies

• Incorporate pressure terms in molecular dynamics force fields

Understanding this conformational transition under pressure would provide fundamental insights into how DNA repair enzymes adapt to extreme environments. The central inactive RNase H domain that serves as a platform for surrounding domains might play a particularly important role in pressure adaptation.

How can researchers differentiate between pressure-specific and temperature-specific adaptations in P. profundum UvrC?

P. profundum is both a piezophile and a psychrophile, requiring careful experimental design:

Matrix experimental approach:

Analysis strategies:

• Two-way ANOVA to differentiate temperature and pressure effects

• Interaction analysis to identify synergistic adaptations

• Comparison with mesophilic piezophiles and psychrophilic non-piezophiles

• Site-directed mutagenesis targeting residues predicted to be involved in either temperature or pressure adaptation

This comprehensive approach would disentangle the complex adaptations that allow P. profundum UvrC to function efficiently in the deep sea. Understanding these adaptations has implications beyond basic science, potentially informing the design of enzymes for biotechnological applications in extreme conditions.

How might P. profundum UvrC interact with other proteins beyond the canonical UvrABC system?

Recent research has revealed unexpected protein interactions in bacterial DNA repair:

Potential non-canonical interactions:

• UvrD helicase: Known to suppress recombination and DNA damage-induced genomic rearrangements

• RecQ helicase: Functions in the same pathway as UvrA to suppress illegitimate recombination

• Cho (UvrC homolog): Provides alternative incision capabilities for certain lesions

Research approaches:

• Protein-protein interaction screening (bacterial two-hybrid, co-immunoprecipitation)

• Genetic interaction mapping (synthetic lethality screening)

• In vitro reconstitution of repair complexes under pressure

• Comparison of interactomes between different P. profundum strains adapted to different pressures

This research would provide a systems-level understanding of how DNA repair pathways are integrated in P. profundum, potentially revealing pressure-specific interaction networks that optimize genomic stability in the deep sea.

What implications do P. profundum UvrC studies have for understanding DNA repair in other extremophiles?

Research on P. profundum UvrC extends beyond this specific organism:

Comparative extremophile research opportunities:

• Compare UvrC from P. profundum (piezophile) with that from Deinococcus radiodurans (radiation-resistant)

• Examine UvrC adaptations across psychrophilic, thermophilic, and halophilic bacteria

• Investigate potential horizontal gene transfer of DNA repair genes among extremophiles

Evolutionary significance:

• Identify convergent adaptations in UvrC across unrelated extremophiles

• Determine if certain structural elements are more conserved in extreme environments

• Assess if extremophile UvrC proteins show greater substrate versatility than mesophilic counterparts

This comparative approach would reveal fundamental principles of protein adaptation to extreme environments while providing insights into the evolution of DNA repair systems across diverse bacterial lineages.