Recombinant Photobacterium profundum Putative Holliday junction resolvase (PBPRA3140)

What is Photobacterium profundum Putative Holliday junction resolvase (PBPRA3140)?

PBPRA3140 is a putative Holliday junction resolvase enzyme (EC 3.1.-.-) derived from the deep-sea bacterium Photobacterium profundum strain SS9. The protein consists of 142 amino acids and functions as an endonuclease that resolves DNA intermediates formed during homologous recombination processes. This enzyme belongs to the RuvC family of resolvases that catalyze the resolution of Holliday junctions during the final stages of homologous recombination. The recombinant form is typically expressed in mammalian cell systems to produce a highly purified protein (>85% by SDS-PAGE) for research applications .

How should recombinant PBPRA3140 be stored for optimal stability?

For optimal stability of recombinant PBPRA3140, the following storage conditions are recommended:

• Short-term storage (up to one week): Store working aliquots at 4°C.

• Long-term storage:

  • Liquid form: Store at -20°C/-80°C for up to 6 months

  • Lyophilized form: Store at -20°C/-80°C for up to 12 months

When preparing the protein for storage, it is advisable to reconstitute it in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 50% before aliquoting and freezing is recommended to prevent damage from freeze-thaw cycles. Repeated freezing and thawing should be avoided as it can lead to protein denaturation and loss of enzymatic activity .

What are the optimal conditions for PBPRA3140 enzymatic activity?

While specific activity conditions for PBPRA3140 are not directly reported in the provided literature, insights can be drawn from related RuvC-family resolvases such as SynRuvC from Synechocystis sp. PCC6803. Based on these related enzymes, the following conditions would likely be optimal for PBPRA3140 activity:

Researchers should empirically determine the optimal conditions specifically for PBPRA3140 through activity assays using synthetic Holliday junctions labeled with fluorescent dyes or radioactive markers to monitor cleavage products .

How does PBPRA3140 compare with other RuvC family resolvases in terms of substrate specificity?

While specific data on PBPRA3140's substrate specificity is limited, comparisons with well-characterized RuvC family resolvases provide valuable insights. RuvC resolvases typically exhibit both structural and sequence specificity:

For experimental determination of PBPRA3140's substrate specificity, researchers should design a panel of DNA substrates including canonical Holliday junctions, replication fork-like structures, and flapped DNA to assess the enzyme's activity profile through gel-based resolution assays.

What biochemical assays are suitable for characterizing PBPRA3140 activity?

Several biochemical assays can be employed to characterize the enzymatic activity of PBPRA3140:

• Holliday Junction Resolution Assay:

  • Synthetic HJ structures labeled with fluorescent dyes or radioactive markers

  • Reaction products analyzed by polyacrylamide gel electrophoresis

  • Quantification of cleaved products to determine kinetic parameters

• DNA Binding Assays:

  • Electrophoretic mobility shift assay (EMSA) to assess binding affinity

  • Fluorescence anisotropy with labeled DNA substrates

  • Surface plasmon resonance (SPR) for real-time binding kinetics

• Metal Ion Dependency Test:

  • Activity assays in the presence of various divalent cations (Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺)

  • Determination of optimal metal ion concentration for catalysis

• Sequence Specificity Mapping:

  • Cleavage site mapping using sequencing gels

  • Next-generation sequencing of cleavage products to identify consensus sequences

• Novel Activity Screening:

  • Testing potential activity on non-canonical substrates such as flapped structures and replication fork intermediates, as observed for SynRuvC

These assays would provide comprehensive characterization of PBPRA3140's enzymatic properties and allow comparison with other Holliday junction resolvases.

How does pressure affect the activity of PBPRA3140 in Photobacterium profundum?

Photobacterium profundum is a piezophilic (pressure-loving) bacterium isolated from deep-sea environments. While specific data on PBPRA3140's pressure response is not provided in the search results, the following considerations are important for researchers studying this enzyme:

• Adaptations to High Pressure:
  P. profundum metabolism responds strongly to pressure regimes, with differences in metabolite pools under varying pressure conditions . As a protein from a piezophilic organism, PBPRA3140 likely possesses structural adaptations that allow function under high hydrostatic pressure.

• Experimental Considerations:
  To study pressure effects on PBPRA3140 activity, researchers should:

  • Use high-pressure bioreactors or specialized equipment to simulate deep-sea conditions

  • Compare enzymatic activity at atmospheric pressure versus elevated pressures (e.g., 40-60 MPa)

  • Analyze structural stability using circular dichroism or differential scanning calorimetry under varying pressure conditions

• Potential Applications:
  Understanding pressure adaptations in PBPRA3140 could provide insights for designing pressure-resistant enzymes for biotechnological applications, particularly in DNA manipulation technologies.

What is the role of PBPRA3140 in DNA repair mechanisms in Photobacterium profundum?

Based on studies of homologous resolvases, PBPRA3140 likely plays several critical roles in DNA repair mechanisms:

• Double-Strand Break Repair:
  Holliday junction resolvases are essential for resolving DNA intermediates formed during homologous recombination repair of double-strand breaks. By analogy to other RuvC proteins, PBPRA3140 likely performs symmetrical cleavage of two sites on opposite strands of Holliday junctions to generate nicked DNA duplexes that can be sealed by ligases .

• Adaptation to Environmental Stress:
  Similar to SynRuvC in cyanobacteria, PBPRA3140 likely contributes to P. profundum's resistance to environmental stressors. Knockdown studies of SynRuvC increased cellular sensitivity to DNA-damaging agents like MMS, HU, and H₂O₂, suggesting a critical role in stress resistance .

• Maintenance of Genome Stability:
  As a piezophilic organism, P. profundum faces unique genotoxic stresses in its deep-sea environment. PBPRA3140 likely contributes to genome stability under these conditions by facilitating efficient repair of DNA damage through homologous recombination pathways.

To experimentally investigate these roles, researchers could employ genetic approaches such as gene knockdown or knockout in P. profundum, followed by assessment of cellular sensitivity to various DNA-damaging agents under different pressure conditions.

What are the key controls for experiments involving recombinant PBPRA3140?

When designing experiments with recombinant PBPRA3140, the following controls should be included:

• Enzyme Activity Controls:

  • Positive control: Well-characterized Holliday junction resolvase (e.g., E. coli RuvC)

  • Negative control: Heat-inactivated PBPRA3140

  • Catalytic site mutant: Create a catalytic residue mutant version as a negative control

• Substrate Controls:

  • Holliday junction without protein

  • Non-junction DNA substrates to confirm specificity

  • Varying DNA sequences to assess sequence preference

• Reaction Condition Controls:

  • Metal ion dependency: Reactions with various divalent cations and EDTA

  • Buffer composition variations

  • Temperature series

• Expression and Purification Controls:

  • SDS-PAGE analysis to confirm protein purity (>85% as indicated in specifications)

  • Western blot verification

  • Mass spectrometry verification of full-length protein

Including these controls will ensure experimental rigor and facilitate proper interpretation of results when working with PBPRA3140.

How can PBPRA3140 be used in comparative studies of Holliday junction resolution mechanisms?

PBPRA3140 presents a valuable tool for comparative studies of Holliday junction resolution mechanisms across different organisms, particularly for understanding adaptations to extreme environments:

• Evolutionary Analysis:

  • Compare sequence and structural features of PBPRA3140 with resolvases from mesophilic organisms

  • Analyze conserved catalytic residues and structural motifs

  • Construct phylogenetic trees to understand evolutionary relationships

• Functional Comparison:

  • Side-by-side activity assays with other RuvC family proteins

  • Analysis of substrate specificity differences

  • Comparison of cofactor requirements and reaction kinetics

• Structural Studies:

  • Comparative crystallography or cryo-EM studies

  • Structure-function relationship analysis

  • Identification of pressure-adaptation features in protein structure

• In vivo Complementation Experiments:

  • Express PBPRA3140 in RuvC-deficient E. coli strains

  • Assess ability to complement DNA repair defects

  • Compare complementation efficiency with other resolvases

Such comparative studies would provide insights into both conserved resolution mechanisms and specialized adaptations of resolvases from extremophilic organisms like P. profundum.

What are common challenges in working with recombinant PBPRA3140 and how can they be addressed?

Researchers working with recombinant PBPRA3140 may encounter several challenges:

For recombinant protein reconstitution, it is recommended to reconstitute PBPRA3140 in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol addition for stability .

How can PBPRA3140 activity be quantified in mixed reaction products?

Quantification of PBPRA3140 activity in complex reaction mixtures requires sensitive and specific analytical methods:

• Gel-Based Quantification:

  • Denaturing polyacrylamide gel electrophoresis of radiolabeled or fluorescently-labeled substrates

  • Phosphorimager or fluorescence scanning for quantification

  • Analysis using densitometry software to calculate percent cleavage

• Real-Time Monitoring:

  • FRET-based assays using dual-labeled Holliday junctions

  • Continuous monitoring of fluorescence changes during resolution

  • Calculation of initial velocities for kinetic parameters

• Chromatographic Methods:

  • HPLC separation of reaction products

  • Use of specific DNA structures as standards

  • Integration of peak areas for quantification

• Next-Generation Sequencing:

  • Deep sequencing of cleavage products

  • Bioinformatic analysis to map cleavage sites

  • Quantification of site preference based on read counts

These methods allow for precise quantification of enzymatic activity and provide insights into substrate preference and reaction kinetics, facilitating comparison with other RuvC family resolvases like SynRuvC .

What are promising areas for future research on PBPRA3140?

Several promising research directions could expand our understanding of PBPRA3140 and its applications:

• Structural Biology:

  • Determination of crystal structure or cryo-EM structure

  • Analysis of pressure-adaptive structural features

  • Understanding of substrate recognition mechanisms

• Pressure Adaptation Studies:

  • Investigation of enzyme kinetics under varying pressure conditions

  • Identification of pressure-sensitive regions in the protein

  • Comparative analysis with resolvases from non-piezophilic organisms

• Application Development:

  • Exploration of PBPRA3140 as a tool for genetic engineering

  • Development of pressure-resistant DNA manipulation enzymes

  • Creation of chimeric resolvases with novel properties

• In Vivo Function:

  • Investigation of PBPRA3140's role in P. profundum stress response

  • Creation of conditional knockdown strains to assess function

  • Global genomic analysis of PBPRA3140 binding sites

• Novel Activities:

  • Exploration of potential flap endonuclease and replication fork intermediate cleavage activities similar to those observed in SynRuvC

  • Testing activity on non-canonical DNA structures

  • Investigation of potential RNA processing capabilities

These research directions would provide comprehensive understanding of PBPRA3140 and potentially lead to novel biotechnological applications.