Recombinant Nitrosomonas europaea UvrABC system protein B (uvrB), partial

What is the functional role of UvrB in the Nucleotide Excision Repair pathway?

UvrB serves as a critical component of the bacterial Nucleotide Excision Repair (NER) system. Within this system, UvrB functions primarily in the damage recognition and verification process. The UvrAB complex first recognizes and binds to distortions in the DNA duplex caused by damage. After recognition, UvrB recruits UvrC to the lesion site, where UvrC acts as a single-stranded DNA endonuclease, cleaving the DNA on both the 5′ and 3′ sides of the lesion. Following this cleavage, UvrD (helicase II) removes the damaged single-stranded segment, allowing DNA polymerase I to synthesize new DNA using the undamaged strand as a template, with ligase completing the repair process .

How does the UvrB protein from Nitrosomonas europaea compare to UvrB proteins in other bacterial species?

While the search results don't provide specific comparative data between N. europaea UvrB and other bacterial UvrB proteins, research on NER systems demonstrates functional conservation across bacterial species. Studies have shown that the Neisseria gonorrhoeae uvrA gene could complement an Escherichia coli uvrA mutant for UV survival, suggesting significant functional similarity between NER components across different bacterial genera . This conservation of function suggests that N. europaea UvrB likely shares core structural and functional features with UvrB proteins from other bacteria, while potentially possessing species-specific adaptations related to N. europaea's ecological niche as an ammonia-oxidizing bacterium.

What forms of recombinant Nitrosomonas europaea UvrB protein are available for research purposes?

Recombinant N. europaea UvrB is available in multiple expression formats to accommodate various experimental needs:

Each recombinant version is typically provided as a lyophilized powder with >85% purity as determined by SDS-PAGE .

What are the optimal storage and reconstitution conditions for recombinant UvrB protein?

For optimal results with recombinant N. europaea UvrB:

• Initial handling: Briefly centrifuge the vial before opening to bring contents to the bottom.

• Reconstitution: Dissolve in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Stabilization: Add glycerol to a final concentration of 5-50% (50% is the standard recommendation).

• Storage: Aliquot the reconstituted protein and store at -20°C/-80°C to avoid repeated freeze-thaw cycles.

• Long-term stability: Properly stored, the protein maintains activity, though specific shelf-life information was not provided in the search results .

How can researchers verify the functional activity of recombinant UvrB in vitro?

To verify UvrB functionality, researchers should consider these methodological approaches:

• DNA binding assays: Measure UvrB's ability to bind damaged DNA in the presence of UvrA using electrophoretic mobility shift assays (EMSAs).

• ATP hydrolysis assays: Since UvrB has ATPase activity stimulated by DNA interaction, monitoring ATP hydrolysis rates can indicate functional activity.

• Functional complementation: As demonstrated with N. gonorrhoeae UvrA complementing E. coli UvrA mutants, similar approaches can test N. europaea UvrB functionality by complementing UvrB-deficient strains .

• Recruitment assays: Assess UvrB's ability to recruit UvrC to damaged DNA sites using purified components.

• UV sensitivity rescue: Test if the recombinant protein can restore UV resistance when introduced into UvrB-deficient bacterial strains .

How can recombinant UvrB be used to study DNA damage recognition mechanisms in Nitrosomonas europaea?

Recombinant UvrB enables sophisticated studies of damage recognition mechanisms through:

• Structure-function analysis: Using site-directed mutagenesis of recombinant UvrB to identify critical residues involved in damage recognition.

• Interaction studies: Employing co-immunoprecipitation or pull-down assays with biotinylated UvrB variants to identify interaction partners in the DNA damage response pathway.

• Substrate specificity determination: Testing UvrB activity on synthetic DNA substrates containing different types of damage to determine recognition specificity.

• Kinetic studies: Measuring the kinetics of UvrB-mediated damage recognition and verification using fluorescently labeled DNA substrates.

• Cryo-EM or crystallography: Using purified recombinant UvrB for structural studies, potentially in complex with DNA and other NER components .

What is the relationship between the NER system and other DNA repair pathways in Nitrosomonas europaea?

Studies with N. gonorrhoeae suggest complex interactions between NER components and other DNA repair systems that may apply to N. europaea:

• Overlap with mismatch repair: UvrD, a helicase in the NER pathway, also plays a role in mismatch repair, as evidenced by higher frequencies of spontaneous mutation in uvrD mutants .

• Independence from recombination pathways: NER mutants demonstrated wild-type levels of DNA transformation and pilin antigenic variation, suggesting NER operates independently from these recombination-dependent processes .

• Interaction with oxidative damage repair: NER mutants showed sensitivity to hydrogen peroxide killing, indicating potential overlap with oxidative damage repair pathways .

• RecA-NER interactions: The effect of RecA expression on UV survival was minor in most uvr mutants but much larger in mfd mutants, suggesting complex pathway interactions .

How might environmental stressors affect UvrB expression and function in Nitrosomonas europaea?

As an ammonia-oxidizing bacterium living in challenging environments, N. europaea likely regulates UvrB expression in response to environmental stressors:

• UV exposure response: Similar to other bacteria, N. europaea likely upregulates UvrB expression following UV exposure to address increased DNA damage.

• Oxidative stress adaptation: Given that NER mutants in N. gonorrhoeae showed sensitivity to hydrogen peroxide, N. europaea UvrB may play a role in responding to oxidative damage from metabolic processes or environmental sources .

• Transcriptional regulation: While specific data on N. europaea UvrB regulation is limited in the search results, expression likely changes during transitions between different environmental conditions, similar to other stress-responsive genes in this organism .

• Nitrosative stress response: As N. europaea produces NO and can experience nitrosative stress, there may be coordinated regulation between nitrogen metabolism genes and DNA repair genes like uvrB .

What experimental design is recommended for studying UvrB-associated DNA repair kinetics?

For robust investigation of UvrB-associated repair kinetics, consider this methodological framework:

• Substrate preparation: Create defined DNA substrates with specific lesions positioned at known locations, possibly fluorescently labeled for real-time monitoring.

• Component assembly: Use purified recombinant components (UvrA, UvrB, UvrC, UvrD) in defined concentrations.

• Reaction conditions optimization:

  • Buffer composition: 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl₂, 1 mM ATP

  • Temperature: 30-37°C (optimize for N. europaea proteins)

  • Timing: Take time points from 0-60 minutes

• Analysis methods:

  • Gel-based assays to visualize incision products

  • Fluorescence-based real-time assays for continuous monitoring

  • Single-molecule approaches for mechanistic insights

• Controls:

  • Undamaged DNA substrate

  • Individual component omission controls

  • Heat-inactivated protein controls

What are the considerations when comparing UvrB function across different bacterial species?

When conducting comparative studies of UvrB across bacterial species, researchers should address:

• Sequence homology analysis: Perform detailed sequence alignment and phylogenetic analysis to identify conserved domains and species-specific variations.

• Expression optimization: Different bacterial UvrB proteins may require tailored expression systems; some may express better in E. coli, others in alternative hosts .

• Functional complementation: Test cross-species complementation by expressing N. europaea UvrB in UvrB-deficient strains of model organisms (e.g., E. coli) to assess functional conservation .

• Biochemical parameter standardization: When comparing kinetic parameters or DNA binding affinity, ensure identical experimental conditions (buffer, temperature, pH) to make valid comparisons.

• Structural considerations: Account for potential differences in protein folding, stability, and post-translational modifications when interpreting functional differences .

How can tagged versions of recombinant UvrB be utilized in advanced research applications?

Recombinant UvrB with various tags enables sophisticated experimental applications:

• Biotinylated Avi-tagged UvrB applications:

  • Pull-down assays for identifying interaction partners

  • Surface immobilization for single-molecule studies

  • Streptavidin-based detection in localization studies

  • Highly specific purification from complex mixtures

• Other potential tag applications:

  • Fluorescent protein fusions for real-time localization

  • Epitope tags for immunoprecipitation studies

  • Affinity tags for one-step purification protocols

  • Split-reporter tags for protein-protein interaction studies in vivo

What are common challenges when working with recombinant UvrB and how can they be addressed?

What methodological adaptations might be necessary when studying UvrB in the context of environmental stress responses?

When investigating UvrB's role in stress responses, consider these methodological adaptations:

• Physiologically relevant conditions: Design experiments that mimic environmental stressors N. europaea encounters:

  • Varying ammonia concentrations

  • Oxygen limitation conditions

  • Presence of nitrite/nitric oxide

  • UV exposure regimens

  • Oxidative stress conditions

• In vivo expression monitoring: Develop reporter systems to monitor uvrB expression under different stress conditions.

• Protein modification analysis: Assess potential post-translational modifications of UvrB that might occur under stress conditions.

• Multi-protein complex analysis: Evaluate how environmental stressors affect UvrB's interactions with other NER components.

• Specialized assays: Develop high-throughput assays to screen multiple stress conditions simultaneously .