Recombinant Photobacterium profundum UvrABC system protein B (uvrB), partial

What is the structure of UvrB in P. profundum and how does it relate to its function?

UvrB in P. profundum, like other bacterial UvrB proteins, contains two domains related in structure to helicases and additional domains unique to repair proteins. The crystal structure contains all elements of an intact helicase, indicating that UvrB utilizes ATP hydrolysis to move along DNA while probing for damage . Analysis of conserved residues and structural comparisons suggest that a flexible β-hairpin is inserted between the two DNA strands to form the tight pre-incision complex with damaged DNA . P. profundum UvrB likely exhibits structural adaptations for function under high hydrostatic pressure (up to 70 MPa) characteristic of its deep-sea habitat .

How does P. profundum UvrB participate in the nucleotide excision repair pathway?

UvrB is a central component of the bacterial NER system, participating in three key processes: damage recognition, strand excision, and repair synthesis . In prokaryotes, UvrA, UvrB, and UvrC orchestrate the recognition and excision of aberrant lesions from DNA . The process begins with UvrA and UvrB forming a damage-sensing complex . After damage recognition, UvrB forms a tight pre-incision complex with the damaged DNA strand, which then facilitates the recruitment of UvrC. UvrC performs incisions on both sides of the lesion, followed by removal of the damaged fragment and repair synthesis . This process in P. profundum is likely adapted to function optimally under the high-pressure conditions of its native deep-sea environment.

What is the cryptic ATPase activity of UvrB and how does it function?

UvrB possesses a cryptic ATPase activity that is essential for DNA repair but is not demonstrable with the purified UvrB protein alone. This activity becomes apparent when UvrB is proteolyzed to yield a 70 kD fragment (UvrB*) that exhibits single-strand DNA dependent ATPase activity . The formation of the UvrAB complex activates this normally sequestered UvrB-associated ATPase . Analyses of nucleotide hydrolysis indicate that this activity is essential to the DNA incision reaction carried out by the UvrABC complex . This cryptic activity reflects a regulatory mechanism where UvrB's helicase-like functionality is controlled to ensure appropriate coordination during the repair process.

What molecular mechanisms enable P. profundum UvrB to function under high pressure?

The molecular mechanisms likely involve several adaptations:

• Protein structural modifications: Alterations in amino acid composition that favor pressure resistance

• Altered hydrophobic packing: Changes in core hydrophobic residues to maintain stability at high pressure

• Modified enzyme kinetics: Adaptations in the ATP binding and hydrolysis mechanisms

Proteomic analysis of P. profundum under different pressure regimes reveals that many proteins are under tight regulation with relatively highly abundant proteins being up- or down-regulated in function of pressure . UvrB might utilize similar regulatory mechanisms to ensure optimal repair activity under varying pressure conditions. Studies on other deep-sea bacteria suggest that pressure adaptations often involve changes in protein flexibility and dynamic properties rather than major structural alterations.

How does pressure affect the UvrA-UvrB damage recognition complex in P. profundum?

The UvrA-UvrB interaction domains are necessary and sufficient for forming the DNA damage-sensing complex of bacterial nucleotide excision repair . In P. profundum, this complex must function efficiently under high pressure. The interface between UvrA and UvrB likely contains specific adaptations that maintain appropriate protein-protein interactions despite the compressive effects of high pressure. Based on studies of cooperative damage recognition, UvrA mutants with decreased DNA binding affinity show reduced rates of DNA transfer to UvrB . In P. profundum, this cooperative mechanism might be enhanced to overcome pressure-induced constraints on protein-DNA interactions.

What expression systems are optimal for producing recombinant P. profundum UvrB?

For recombinant expression of P. profundum proteins, E. coli-based expression systems have been successfully employed. The pTYB1 expression system has been used for similar proteins , which creates a fusion protein with an intein tag for easy purification. For P. profundum UvrB specifically, expression conditions should account for its psychrophilic nature by using lower induction temperatures (15-20°C) to ensure proper folding. The expression protocol should include:

• Transformation of the expression construct into a suitable E. coli strain (BL21(DE3) or similar)

• Culture at reduced temperature (15-20°C) following induction

• Addition of appropriate cofactors (ATP, Mg²⁺) in the purification buffers to stabilize the protein

• Verification of protein activity under varying pressure conditions

Consideration should be given to codon optimization for the expression host, as P. profundum may have different codon usage patterns compared to E. coli.

How can the activity of recombinant P. profundum UvrB be assayed under different pressure conditions?

Activity assays for P. profundum UvrB should evaluate both its ATPase activity and DNA repair function under varying pressure conditions. Methodologies include:

• ATPase activity measurement: Using coupled enzyme assays that measure the release of inorganic phosphate or ADP formation at different pressures . The rate of ATP hydrolysis can be calculated from the linear change in absorbance at 340 nm using a spectrophotometer adapted for high-pressure measurements.

• Oligonucleotide incision assay: Using a 5′ end-labeled duplex DNA containing a site-specific lesion (e.g., fluorescein-adducted thymine) and monitoring incision products using denaturing polyacrylamide gel electrophoresis . This assay can be performed following pressure treatment of the UvrABC proteins.

• DNA binding assays: Electrophoretic mobility shift assays (EMSA) can assess DNA binding capacity under different pressure regimes by analyzing samples immediately after pressure treatment .

• High-pressure microscopic chamber: Direct observation of protein-DNA interactions under pressure, similar to the chambers used for motility studies of P. profundum SS9 that maintained motility up to 150 MPa .

What high-pressure cultivation methods are suitable for P. profundum growth prior to UvrB isolation?

P. profundum requires specialized cultivation techniques for growth under high pressure. Effective cultivation methods include:

• Pressure vessel cultivation: Growth in sealed containers (e.g., Pasteur pipettes) within water-cooled pressure vessels capable of maintaining 28 MPa at 17°C .

• Anaerobic conditions: Cultures should be grown anaerobically in marine broth supplemented with glucose and HEPES buffer (pH 7.5) .

• Temperature control: Maintaining optimal growth temperature (15-17°C) during high-pressure cultivation .

• Culture vessel preparation: Using sealed plastic Pasteur pipettes containing 6 mL of culture with air excluded to ensure even hydrostatic pressure distribution and anaerobic conditions .

• Growth monitoring: Allowing cultures to reach stationary phase (approximately 5 days) before harvesting by centrifugation at 800×g for 10 minutes .

These methods ensure that P. profundum expresses proteins in their pressure-adapted conformation, which may be critical for understanding the native structure and function of UvrB.

How does the P. profundum UvrABC system recognize DNA damage in high-pressure environments?

DNA damage recognition by the UvrABC system is a multi-step process involving cooperative action between UvrA and UvrB. In bacterial systems:

• UvrA, as part of an UvrA₂B or UvrA₂B₂ complex, first scans DNA for altered structures .

• Upon damage detection, UvrA transfers the DNA to UvrB, forming a tight UvrB-DNA pre-incision complex .

• UvrB's β-hairpin structure is inserted between the two DNA strands at the damage site .

• This conformation change signals for UvrC recruitment and subsequent incision .

In P. profundum, this mechanism likely includes adaptations for high-pressure environments. The cooperative nature of the UvrA-UvrB interaction helps overcome individual protein limitations. Studies with UvrA mutants show that even proteins with reduced DNA binding affinity can efficiently transfer DNA to UvrB once the UvrA₂B complex is formed . This cooperative mechanism may be particularly important in high-pressure environments where protein-DNA interactions might be altered.

What role does ATP hydrolysis play in P. profundum UvrB function during DNA repair?

ATP hydrolysis is essential for UvrB function in DNA repair. The mechanism involves:

• ATP binding to UvrB, which facilitates structural changes necessary for DNA interaction .

• The cryptic ATPase activity of UvrB that becomes activated upon interaction with UvrA and DNA .

• ATP hydrolysis that drives conformational changes in UvrB, allowing it to move along DNA and probe for damage .

• Energy from ATP hydrolysis that powers the formation of the stable pre-incision complex .

For P. profundum UvrB, this ATP-dependent mechanism might be specially adapted to function efficiently under high pressure. Research on UvrB from other bacteria shows that domain 4 of UvrB acts as an autoinhibitory domain for both DNA binding and ATPase activity, which becomes activated through interaction with UvrA . This regulatory mechanism ensures that ATP hydrolysis occurs only when appropriate, which may be particularly important in the energy-limited deep-sea environment.

How does UvrB coordinate with UvrA and UvrC in the complete nucleotide excision repair pathway?

The coordination between UvrA, UvrB, and UvrC involves a precisely orchestrated sequence:

• Initial complex formation: UvrA and UvrB form a UvrA₂B or UvrA₂B₂ complex that scans DNA for damage .

• Damage verification: Upon encountering a lesion, UvrB verifies the damage using its cryptic ATPase activity and β-hairpin structure .

• Handoff mechanism: UvrA transfers the damaged DNA to UvrB and dissociates, leaving UvrB bound at the damage site .

• UvrC recruitment: UvrB adopts a conformation that signals for UvrC binding .

• Dual incision: UvrC makes incisions at the 3′ and 5′ sides of the damage .

• Repair completion: UvrD helicase and DNA polymerase I remove the damaged oligonucleotide and synthesize new DNA, with DNA ligase sealing the nick .

In P. profundum, this coordination must function under high pressure. The interfaces between these proteins likely contain specific adaptations to maintain appropriate interactions despite the compressive effects of high pressure. The protein-protein interfaces between UvrA-UvrB and UvrB-UvrC are potential sites for pressure-specific adaptations in the P. profundum repair system.

What genomic features distinguish P. profundum UvrB from homologs in non-piezophilic bacteria?

The genomic analysis of P. profundum strains reveals several features that likely influence UvrB function:

• Strain-specific adaptations: Multiple strains of P. profundum have been isolated from different ocean depths and display remarkable differences in their physiological responses to pressure . These differences extend to genomic features that correlate to environmental differences and define the ecological niche of each strain .

• Genome plasticity: Variations in gene content and specific gene sequences under positive selection contribute to adaptation to different pressure environments . UvrB sequences from deep-sea versus shallow-water strains likely show specific amino acid substitutions that optimize function at different pressures.

• Pressure-responsive regulation: Similar to other genes in P. profundum, uvrB expression may be regulated in response to pressure changes. Several stress response genes (htpG, dnaK, dnaJ, and groEL) are upregulated in response to atmospheric pressure in strain SS9 , suggesting that DNA repair systems might be similarly regulated.

• Evolutionary relationship: P. profundum is closely related to the genus Vibrio based on 16S rRNA sequences , suggesting that its UvrB may share core features with Vibrio species while possessing specific adaptations for the deep-sea environment.

How does natural selection shape UvrB function in deep-sea versus shallow-water P. profundum strains?

The comparison between deep-sea and shallow-water P. profundum strains provides insights into selective pressures on UvrB:

• Pressure adaptation gradient: P. profundum strain SS9 (isolated from 2.5 km depth) grows optimally at 28 MPa, while strain 3TCK (isolated from San Diego Bay) grows optimally at 0.1 MPa . These differences likely reflect selection for pressure-specific adaptations in proteins including UvrB.

• Genetic modifications: Analysis of the sequenced genome of P. profundum strain 3TCK reveals unique genomic features that correlate to environmental differences compared to the deep-sea piezopsychrophilic strain SS9 . These genomic differences likely extend to genes involved in DNA repair pathways.

• Functional trade-offs: Adaptations that optimize UvrB function at high pressure may compromise its performance at atmospheric pressure, creating distinct evolutionary trajectories for deep-sea versus shallow-water strains.

• Ecological niche specialization: The Hutchinsonian niche of each strain is defined by genomic features adapted to specific environmental conditions , suggesting that UvrB function has been optimized for the prevalent DNA damage types and repair requirements of each environment.

Can P. profundum UvrB complement UvrB-deficient strains of model organisms like E. coli?

While direct complementation studies with P. profundum UvrB are not described in the search results, several factors would influence its ability to complement UvrB-deficient model organisms:

• Functional conservation: The basic mechanism of UvrABC-mediated repair is conserved across bacterial species, suggesting that P. profundum UvrB might functionally replace E. coli UvrB.

• Temperature constraints: P. profundum is psychrophilic, growing optimally at 15°C , which may limit the functionality of its proteins at the higher temperatures used for E. coli cultivation (37°C).

• Pressure adaptations: P. profundum UvrB may contain structural features optimized for high-pressure environments that could affect its performance at atmospheric pressure.

• Protein-protein interactions: Successful complementation would require P. profundum UvrB to interact properly with E. coli UvrA and UvrC. Studies on UvrA mutants show that decreased UvrA DNA binding can be compensated by its ability to efficiently transfer DNA to UvrB , suggesting that the UvrA-UvrB interface is critical for proper function.

• Expression regulation: The expression mechanisms controlling P. profundum uvrB may differ from those in E. coli, potentially affecting complementation efficiency.

Complementation studies could provide valuable insights into the functional conservation and specialization of the UvrB protein across different bacterial species and environmental conditions.

How can insights from P. profundum UvrB contribute to our understanding of DNA repair in extreme environments?

P. profundum UvrB offers unique insights into DNA repair adaptations:

• Pressure-adapted mechanisms: Understanding how P. profundum UvrB functions under high pressure can reveal general principles of protein adaptation to extreme conditions. Pressure affects protein structure, dynamics, and interactions in ways distinct from other stressors like temperature or pH.

• Environmental damage specificity: Deep-sea environments present unique DNA damage profiles (radiation patterns, chemical exposures, pressure effects on DNA structure) that may select for specialized repair mechanisms.

• Energy efficiency: Deep-sea environments are often energy-limited, potentially selecting for repair systems that optimize energy utilization. The ATPase activity of UvrB might be specially regulated in P. profundum to minimize energy expenditure while maintaining repair efficiency.

• Structural flexibility vs. stability: High pressure generally favors compact protein conformations, yet DNA repair requires significant conformational changes. P. profundum UvrB likely represents an evolutionary solution to this paradox.

• Cooperative mechanisms: The ability of UvrA and UvrB to work cooperatively in damage recognition may be particularly important in extreme environments where individual protein functions might be compromised.

What methodological approaches can be used to study UvrB function under pressure in laboratory settings?

Several methodological approaches can be employed:

• High-pressure biochemical assays: Using pressure chambers adapted for spectrophotometric measurements to assess ATPase activity and DNA binding under pressure in real-time.

• Pressure-jump experiments: Rapidly changing pressure while monitoring protein conformation or activity to detect pressure-dependent transitions.

• High-pressure structural biology: X-ray crystallography or NMR studies of P. profundum UvrB under pressure to identify conformational changes.

• Comparative mutagenesis: Creating chimeric proteins with domains from pressure-adapted and non-adapted UvrB proteins to identify critical regions for pressure adaptation.

• In vivo repair assays: Measuring DNA repair efficiency in P. profundum under varying pressure conditions using methods like the in vivo photoreactivation assay described for P. profundum .

• High-pressure microscopy: Direct visualization of fluorescently labeled UvrB interacting with damaged DNA under pressure, similar to techniques used for observing P. profundum motility under pressure .

How might studying P. profundum UvrB inform synthetic biology approaches to creating pressure-resistant organisms?

Research on P. profundum UvrB could inform synthetic biology in several ways:

• Design principles: Identifying specific amino acid substitutions or structural features that confer pressure resistance could provide design principles for engineering pressure-resistant proteins.

• Modular adaptation: Understanding which domains of UvrB are most critical for pressure adaptation could allow for modular replacement in synthetic systems.

• Regulatory circuits: Deciphering how P. profundum regulates UvrB expression in response to pressure could inform the design of pressure-responsive genetic circuits.

• Minimal adaptations: Determining the minimal set of modifications needed for pressure adaptation could streamline engineering efforts.

• Cross-environmental functionality: Understanding how P. profundum maintains repair activity across its wide pressure range (0.1-90 MPa) could inform the design of broadly functional synthetic systems.

Such applications could be valuable for developing microorganisms for bioremediation in deep-sea environments, biotechnology processes under high pressure, or even organisms for extraterrestrial environments with different pressure regimes.

What advantages does P. profundum offer as a model system for studying pressure adaptation of DNA repair?

P. profundum has several advantages as a model organism:

• Cultivation flexibility: Unlike many piezophiles, P. profundum can grow at both atmospheric pressure and high pressure (up to 90 MPa) , facilitating laboratory cultivation and genetic manipulation.

• Genetic tools: The ability to grow at atmospheric pressure allows for standard genetic manipulation techniques to be applied .

• Strain diversity: Multiple strains isolated from different depths with varying pressure optima provide natural comparative systems . The strain SS9 (optimal growth at 28 MPa) versus strain 3TCK (optimal growth at 0.1 MPa) comparison is particularly valuable .

• Genome sequence availability: The sequenced genomes of P. profundum strains SS9 and 3TCK allow for genomic and proteomic analyses .

• Physiological studies: Established protocols for studying gene expression, protein function, and cellular processes under different pressure conditions .

• Related to model organisms: Close relationship to Vibrio species facilitates comparative genomics and functional studies .

These features make P. profundum an excellent model for studying how fundamental cellular processes, including DNA repair, adapt to function under varying pressure conditions.

How do different P. profundum strains vary in their UvrB function and regulation?

While the search results don't directly address UvrB variation between strains, insights can be drawn from strain differences:

• Genomic variations: The genome sequence of P. profundum strain 3TCK (a non-piezophilic strain) differs from strain SS9 (piezopsychrophilic), with variations in gene content and specific gene sequences under positive selection . These differences likely extend to the UvrB gene and its regulation.

• Pressure optima: Strain SS9 has optimal growth at 15°C and 28 MPa, while strain 3TCK grows optimally at 9°C and 0.1 MPa . These physiological differences suggest corresponding adaptations in DNA repair systems.

• Expression regulation: Similar to flagellar genes where differential expression is observed between pressure conditions , UvrB expression likely varies between strains adapted to different pressures.

• Functional adaptation: The UvrB protein from high-pressure-adapted strains likely contains amino acid substitutions that optimize its ATPase activity, DNA binding, and protein-protein interactions under high pressure.

• Repair efficiency: Deep-sea strains may exhibit enhanced repair efficiency for the types of DNA damage prevalent in their environment, possibly showing specialized recognition of pressure-induced DNA structural alterations.

Comparative studies of UvrB from different P. profundum strains could reveal the molecular basis of pressure adaptation in DNA repair systems.

What research techniques have been most successful in studying P. profundum proteins under pressure?

Several successful techniques for studying P. profundum under pressure emerge from the search results:

• Pressure vessel cultivation: Growing cultures in sealed containers within water-cooled pressure vessels at controlled temperature and pressure .

• Label-free proteomic analysis: Shotgun proteomic analysis using mass spectrometry to identify differentially expressed proteins under different pressure conditions .

• High-pressure microscopic chambers: Direct observation of cellular processes (such as motility) under varying pressure conditions up to 150 MPa .

• Comparative genomics: Analysis of genomic differences between strains adapted to different pressure environments .

• In vivo functional assays: Methods like the photoreactivation assay that can assess repair function under different conditions .

• Genetic manipulation: Creating deletion constructs and tracking their effects on function under different pressure conditions .

• Expression analysis: Monitoring gene expression changes in response to pressure variations .

These techniques provide a comprehensive toolkit for investigating the structural and functional properties of P. profundum proteins, including UvrB, under varying pressure conditions.

What unresolved questions remain about P. profundum UvrB function under pressure?

Several important questions remain unresolved:

• Structural adaptations: What specific amino acid substitutions or structural features enable P. profundum UvrB to function under high pressure?

• Pressure thresholds: At what pressure does P. profundum UvrB function begin to decline, and what aspects of its activity are most pressure-sensitive?

• Damage specificity: Does P. profundum UvrB show enhanced recognition of specific types of DNA damage that might be more prevalent in the deep sea?

• Protein-protein interactions: How are the interfaces between UvrB and its partners (UvrA, UvrC) adapted to maintain function under pressure?

• Energy efficiency: How is the ATPase activity of UvrB optimized for the energy-limited deep-sea environment?

• Regulatory networks: How is UvrB expression regulated in response to pressure changes, and what signaling pathways are involved?

• Horizontal gene transfer: Has the UvrB gene in P. profundum acquired adaptations through horizontal gene transfer from other piezophiles?

• Repair pathway integration: How does the NER pathway interact with other DNA repair pathways under high-pressure conditions?

Addressing these questions would significantly advance our understanding of DNA repair adaptation to extreme environments.

How might advances in structural biology techniques contribute to understanding P. profundum UvrB?

Advanced structural biology techniques could provide crucial insights:

• Cryo-electron microscopy: Revealing the structure of the complete UvrABC complex in action, potentially capturing the dynamic conformational changes during the repair process.

• High-pressure X-ray crystallography: Determining the structural changes in UvrB under different pressure conditions.

• High-pressure NMR: Examining the dynamics and conformational changes of UvrB domains under pressure.

• Hydrogen-deuterium exchange mass spectrometry: Identifying regions of UvrB with pressure-dependent changes in solvent accessibility or dynamics.

• Single-molecule FRET: Observing the conformational dynamics of UvrB during damage recognition and verification under pressure.

• Molecular dynamics simulations: Modeling the effects of pressure on UvrB structure and interactions, particularly in regions identified as important for function.

• AlphaFold or similar AI-based prediction tools: Predicting structural adaptations in P. profundum UvrB compared to mesophilic homologs.

These approaches could reveal the molecular basis of UvrB adaptation to high pressure and inform the design of pressure-resistant proteins for biotechnology applications.

What implications do pressure adaptations in DNA repair systems have for astrobiology and the search for extraterrestrial life?

Pressure adaptations in DNA repair systems have significant astrobiological implications:

• Subsurface ocean worlds: Icy moons like Europa and Enceladus harbor subsurface oceans under high pressure, making pressure-adapted DNA repair mechanisms potentially relevant for life in these environments.

• Evolutionary convergence: Understanding whether pressure adaptations in DNA repair follow predictable patterns could inform expectations for extraterrestrial life.

• Extremophile capabilities: P. profundum's ability to function across a wide pressure range (0.1-90 MPa) demonstrates the adaptability of life, suggesting that DNA repair systems might similarly adapt to conditions on other worlds.

• Biomarker identification: Pressure-adapted proteins like UvrB might serve as biomarkers for life in high-pressure environments.

• Planetary protection: Understanding how terrestrial microbes with pressure-adapted repair systems might survive in extraterrestrial environments has implications for planetary protection policies.

• Life detection experiments: Knowledge of how pressure affects DNA repair could inform the design of life detection experiments for high-pressure environments.

• Origin of life scenarios: Insights into pressure adaptation mechanisms might contribute to theories about life's origins in deep-sea hydrothermal vents on Earth or similar environments elsewhere.