Recombinant Vibrio vulnificus UvrABC system protein A (uvrA), partial

What is UvrABC system protein A (uvrA) in Vibrio vulnificus?

UvrA is a critical component of the UvrABC nucleotide excision repair (NER) system in V. vulnificus. It functions as an ATPase and DNA-binding protein that initiates the recognition of DNA lesions. In V. vulnificus strain CMCP6, UvrA consists of 940 amino acids with a molecular mass of approximately 104 kDa, belonging to the ABC transporter superfamily . The protein forms a damage recognition complex with UvrB that scans DNA for abnormalities, acting as the first responder in the bacterial NER pathway .

What is the primary function of UvrA in DNA repair mechanisms?

UvrA initiates the bacterial nucleotide excision repair pathway through damage recognition and verification. Specifically, it forms a heterotetrameric complex (UvrA₂B₂) that scans the DNA for lesions. Upon binding to a potential damaged site, the DNA wraps around one UvrB monomer in an ATP-dependent manner, which causes local melting of the DNA helix and facilitates the insertion of UvrB's β-hairpin between DNA strands . UvrA then probes one strand for lesions. If damage is confirmed, the UvrA subunits dissociate, leaving the UvrB-DNA preincision complex that subsequently recruits UvrC, releasing the second UvrB. If no lesion is found, the complex continues scanning, checking the other strand for damage .

What expression systems are effective for producing recombinant V. vulnificus UvrA protein?

Multiple expression systems have been successfully employed for UvrA production:

For most research applications, E. coli-based expression (as seen in product CSB-EP762668VCQ) is sufficient and provides the best yield-to-cost ratio for UvrA expression . When expressing in E. coli, incorporating an Avi-tag for biotinylation (as in CSB-EP762668VCQ-B) can significantly enhance protein detection and immobilization capabilities .

What purification strategies yield the highest purity for UvrA protein?

For obtaining >85% purity (as achieved in commercial preparations), a multi-step purification approach is recommended :

• Initial capture: Nickel or cobalt affinity chromatography for His-tagged constructs

• Intermediate purification: Ion exchange chromatography (typically anion exchange as UvrA has a theoretical pI of 5.8-6.2)

• Polishing step: Size exclusion chromatography to remove aggregates and degradation products

• Quality control: SDS-PAGE and Western blot analysis to confirm purity and identity

The choice of buffer systems is critical - phosphate buffers with 5-10% glycerol and reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) enhance stability during purification .

How can recombinant UvrA be used to study DNA damage recognition mechanisms in vitro?

Methodological approach for investigating UvrA-DNA interactions:

• Electrophoretic Mobility Shift Assays (EMSA): Use purified UvrA protein with various DNA substrates containing specific lesions (e.g., UV photoproducts, chemical adducts). Characterize binding affinity and specificity through titration experiments.

• Surface Plasmon Resonance (SPR): Immobilize DNA substrates on sensor chips and measure real-time association/dissociation kinetics of UvrA binding, with and without ATP.

• Fluorescence Anisotropy: Label DNA substrates with fluorophores and monitor changes in anisotropy upon UvrA binding to determine binding constants and cooperative effects.

• ATP Hydrolysis Assays: Couple UvrA ATPase activity measurements with DNA binding using malachite green or NADH-coupled assays to correlate ATP consumption with damage recognition steps.

These approaches have revealed that UvrA employs ATP-dependent conformational changes during damage verification, with distinct binding modes for different DNA lesions .

What role does UvrA play in V. vulnificus virulence and stress adaptation?

Recent genomic analyses of evolved V. vulnificus variants revealed that mutations in DNA repair systems, including UvrA, are associated with adaptation to environmental stresses . While UvrA's primary function is in DNA repair, its indirect contribution to pathogenesis appears to be through:

• Survival under oxidative stress: UvrA helps repair DNA damage caused by reactive oxygen species produced during host immune responses. Strains with intact UvrABC systems show enhanced survival in phagocytes.

• Adaptation to UV exposure: In environmental settings, UvrA facilitates survival under solar radiation, potentially contributing to persistence in aquatic reservoirs.

• Stress-induced genomic evolution: During infection and environmental transitions, UvrA activity may influence the rate and spectrum of mutations, impacting the emergence of variants with altered virulence properties.

Genomic analyses of evolved V. vulnificus strains have identified UvrA among the proteins that undergo mutation during adaptation to new environments, suggesting its role in the bacterium's capacity to persist under changing conditions .

How does V. vulnificus UvrA differ from homologs in other bacterial species?

Comparison of UvrA proteins across Vibrio species and other bacteria reveals important evolutionary adaptations:

The regions showing highest conservation correspond to ATP-binding cassettes and UvrB interaction interfaces, while DNA-binding domains show greater variation. This suggests species-specific adaptation in DNA damage recognition while maintaining core repair functionality.

What structural features of UvrA are critical for its function in DNA repair?

UvrA contains several key structural elements essential for its function:

• ATP-binding cassettes: Two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP to drive conformational changes required for damage recognition.

• Zinc finger motifs: Coordinate zinc ions and mediate protein-DNA interactions; critical for specific recognition of damaged DNA.

• Insertion domains: Located between the ATP-binding cassettes, these regions determine the specificity of DNA damage recognition.

• UvrB interaction surfaces: Regions that facilitate the formation of the UvrA₂B₂ heterotetrameric complex.

• Signature sequence: Characteristic motif of ABC ATPases required for ATP binding and hydrolysis.

Structural studies have shown that UvrA undergoes significant conformational changes between ATP-bound and ADP-bound states, which are essential for its "molecular matchmaker" function in delivering UvrB to damaged DNA sites .

What mutagenesis strategies can reveal structure-function relationships in UvrA?

To elucidate critical functional domains in UvrA, implement these targeted approaches:

These approaches, combined with functional assays measuring DNA binding, ATPase activity, and complex formation with UvrB, can provide comprehensive insights into UvrA's mechanism of action .

How can protein-protein interaction studies reveal UvrA's role in the repair complex?

To characterize UvrA's interactions with other repair proteins:

• Co-immunoprecipitation (Co-IP): Use anti-UvrA antibodies to pull down complexes from V. vulnificus lysates, followed by mass spectrometry to identify interacting partners.

• Bacterial two-hybrid analysis: Screen for protein-protein interactions between UvrA and other V. vulnificus proteins to identify novel binding partners beyond the known UvrB interaction.

• Isothermal titration calorimetry (ITC): Measure thermodynamic parameters of UvrA-UvrB interactions using purified proteins to determine binding affinity and stoichiometry.

• Förster resonance energy transfer (FRET): Label UvrA and UvrB with appropriate fluorophores to monitor real-time complex formation and conformational changes during damage recognition.

• Crosslinking coupled with mass spectrometry: Identify specific residues involved in UvrA-UvrB interactions to map the interaction interface at amino acid resolution.

These approaches have revealed that UvrA interacts not only with UvrB but potentially with other cellular components involved in DNA repair and stress response pathways .

How does V. vulnificus modulate UvrA expression under different environmental conditions?

V. vulnificus encounters diverse stresses during infection and environmental persistence that affect UvrA expression:

• Temperature fluctuations: UvrA expression increases at temperatures associated with human infection (37°C) compared to environmental temperatures (20-25°C).

• Oxidative stress: Exposure to reactive oxygen species upregulates UvrA as part of the SOS response.

• UV radiation: Environmental UV exposure strongly induces UvrA expression through LexA-dependent regulation.

• Growth phase dependency: UvrA levels vary with growth phase, with highest expression typically during exponential growth.

• Iron availability: Iron limitation conditions, similar to those encountered during infection, may alter UvrA expression patterns.

Quantitative PCR studies comparing UvrA expression across these conditions reveal complex regulatory networks integrating environmental signals with DNA repair capacity .

How does UvrA contribute to antibiotic resistance mechanisms?

UvrA's role in antibiotic resistance appears to be multifaceted:

• Direct repair of DNA damage: Some antibiotics (e.g., fluoroquinolones) directly damage DNA, and UvrA-initiated repair mitigates this damage.

• Stress-induced mutagenesis: Under antibiotic stress, UvrA activity may influence mutation rates, potentially accelerating the acquisition of resistance mutations.

• Cross-talk with stress response pathways: UvrA function intersects with general stress responses, including those activated by antibiotics that do not directly damage DNA.

• Biofilm formation: DNA repair systems including UvrA have been implicated in biofilm development, which enhances antibiotic tolerance.

Studies with UvrA-deficient strains show increased sensitivity to several antibiotics, suggesting that targeting UvrA or other NER components could potentially enhance antimicrobial efficacy against V. vulnificus infections .

What emerging technologies can advance our understanding of UvrA function?

Several cutting-edge approaches hold promise for UvrA research:

• Cryo-electron microscopy: For high-resolution structural analysis of UvrA in complex with DNA and UvrB, capturing transient intermediates in the damage recognition process.

• Single-molecule FRET: To observe real-time conformational changes in individual UvrA molecules during ATP binding/hydrolysis and damage recognition.

• Nanopore technology: For studying UvrA-DNA interactions at single-molecule resolution by monitoring changes in current as UvrA binds to DNA threaded through nanopores.

• DNA curtains: To visualize UvrA scanning along DNA molecules and identify differences in behavior at damaged versus undamaged sites.

• CRISPR-Cas9 genome editing: For generating precise mutations in the chromosomal UvrA gene to study phenotypic effects in the native genetic context.

These technologies can overcome limitations of traditional biochemical approaches by providing dynamic, real-time information about UvrA function at unprecedented resolution .

How might UvrA serve as a target for novel antimicrobial strategies?

Targeting UvrA as an antimicrobial strategy presents several promising approaches:

• Inhibitors of UvrA ATPase activity: Small molecules that interfere with ATP binding or hydrolysis could disrupt the damage recognition process.

• Disruptors of UvrA-UvrB interaction: Peptides or small molecules that prevent complex formation would inhibit the initial steps of nucleotide excision repair.

• DNA mimetics: Molecules that bind to UvrA's DNA-binding domain could sequester the protein away from genuine DNA damage sites.

• Adjuvants for existing antibiotics: UvrA inhibitors could potentially sensitize V. vulnificus to antibiotics that cause DNA damage.

• Targeted protein degradation: Proteolysis-targeting chimeras (PROTACs) directed against UvrA could reduce cellular levels of the protein.

While these approaches remain theoretical for V. vulnificus, they represent rational strategies based on the protein's critical role in DNA repair and bacterial survival under stress conditions .