Recombinant Chlamydophila caviae Holliday junction ATP-dependent DNA helicase RuvA (ruvA)

Basic Research Questions

• What is the functional role of RuvA in Chlamydophila caviae DNA recombination?

  RuvA in C. caviae plays a crucial role in homologous recombination, which generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks. Specifically, RuvA is involved in the branch migration of Holliday junctions, which are key intermediates in the recombination process. RuvA functions by binding to Holliday junctions and facilitating the loading of RuvB, a hexameric DNA helicase, onto the junction . This RuvA-RuvB complex drives branch migration by passing DNA through the central cavity of the RuvB rings. In C. caviae, as in other Chlamydiaceae, the ruvA gene is conserved and encodes a protein that likely functions similarly to its E. coli counterpart, as part of the DNA recombination machinery necessary for genetic exchange and DNA repair .

• How does the RuvABC complex function collectively in DNA repair mechanisms?

  The RuvABC complex operates as a coordinated machinery for Holliday junction processing:

  1. RuvA binds specifically to Holliday junctions, imposing a 4-fold symmetric square-planar structure on the DNA

  2. RuvB, an ATP-dependent helicase, forms hexameric rings that are loaded onto the DNA by RuvA

  3. RuvC, a homodimeric endonuclease, recognizes and cleaves specific sequences at the Holliday junction

  Together, these proteins facilitate branch migration and resolution of Holliday junctions via a concerted enzymatic mechanism. RuvB provides the motor force for branch migration, requiring ATP hydrolysis, while RuvA provides specificity by binding to the junction, reducing the requirement for RuvB by 50-fold . RuvC then resolves the migrated junction by introducing symmetrically related nicks in two strands of like polarity . This coordinated action ensures efficient processing of recombination intermediates into mature recombinants, crucial for DNA repair following damage such as UV irradiation .

• What structural features characterize RuvA and how do they relate to its function?

  RuvA has a specific structure optimized for Holliday junction binding and RuvB interaction. In E. coli, which provides a model for understanding C. caviae RuvA, the protein forms tetramers that bind the junction and impose a 4-fold symmetric square-planar structure on the DNA . This structural arrangement is critical for proper function, as it:

  • Opens up the junction to prevent branch migration reversal

  • Creates binding sites for RuvB hexamers

  • Positions the DNA strands for efficient passage through RuvB rings

  The tetramer structure of RuvA, with its ability to bind specifically to Holliday junctions, ensures that RuvB is properly targeted to these recombination intermediates. Studies in E. coli have shown that RuvA-RuvB interactions are crucial for efficient branch migration . While the specific structure of C. caviae RuvA has not been as extensively characterized as its E. coli counterpart, genome analysis indicates conservation of the functional domains necessary for these interactions .

Intermediate Research Questions

• What methods are most effective for expressing and purifying recombinant C. caviae RuvA protein?

  Based on established protocols for related proteins, the following methodology is recommended for expressing and purifying recombinant C. caviae RuvA:

  Expression System Options:

  • E. coli BL21(DE3) with pET-based expression vectors has proven effective for expressing chlamydial proteins

  • Yeast or baculovirus systems may be considered for proteins that misfold in E. coli

  Purification Protocol:

  1. Clone the C. caviae ruvA gene into an expression vector with an appropriate tag (His6 or GST)

  2. Transform into E. coli and induce expression with IPTG (0.5-1 mM)

  3. Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM DTT

  4. Purify using affinity chromatography (Ni-NTA for His-tagged proteins)

  5. Further purify by ion-exchange chromatography followed by gel filtration

  Critical Considerations:

  • Include protease inhibitors during purification to prevent degradation

  • Optimize temperature (typically 25-30°C rather than 37°C) to enhance solubility

  • Consider co-expression with chaperones if solubility issues arise

  • Verify protein activity using in vitro Holliday junction binding assays

• How can researchers effectively design Holliday junction substrates for in vitro studies of C. caviae RuvA activity?

  For studying C. caviae RuvA activity in vitro, carefully designed Holliday junction substrates are essential. Based on established methodologies:

  Synthetic Holliday Junction Design:

  1. Design four oligonucleotides (50-80 nucleotides each) that can anneal to form a cruciform structure

  2. Include regions of homology where branch migration can occur

  3. Consider incorporating fluorescent labels (e.g., Cy3, Cy5) at strategic positions for FRET-based assays

  4. For branch migration assays, design junctions with heterologous arms to create a substrate that can be visualized during migration

  Assembly Protocol:

  1. Mix equimolar amounts of the four oligonucleotides in annealing buffer (e.g., 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl₂)

  2. Heat to 95°C for 5 minutes

  3. Cool slowly to room temperature (1°C/minute) to ensure proper annealing

  4. Purify assembled junctions by native PAGE

  Activity Assays:

  • Branch migration assays require ATP (1-5 mM) and MgCl₂ (10 mM)

  • Junction binding can be assessed by electrophoretic mobility shift assays (EMSA)

  • For studying RuvABC complex formation, include purified RuvB and RuvC proteins

• What are the optimal conditions for studying RuvA-RuvB interactions in C. caviae?

  To effectively study RuvA-RuvB interactions in C. caviae, researchers should consider the following conditions based on studies of E. coli RuvAB complexes:

  Buffer Composition:

  • 20 mM Tris-HCl (pH 7.5)

  • 10 mM MgCl₂ (essential for ATP hydrolysis)

  • 1 mM DTT (to maintain reducing conditions)

  • 50-100 mM NaCl (higher concentrations may impair complex formation)

  Reaction Requirements:

  • ATP is absolutely required (1-5 mM) for RuvB activity

  • Protein concentrations: Use at least a 2:1 molar ratio of RuvB to RuvA (reflecting the hexameric RuvB to tetrameric RuvA stoichiometry)

  • Temperature: 37°C is optimal for branch migration activity

  Assay Options:

  1. Biochemical assays: ATP hydrolysis assays using malachite green or coupled enzyme systems

  2. Branch migration assays: Using synthetic Holliday junctions with differentially labeled arms

  3. Physical interaction studies: Co-immunoprecipitation, analytical ultracentrifugation, or surface plasmon resonance

  4. Cross-linking experiments: To capture transient interactions between RuvA and RuvB

  Since RuvA from C. caviae has not been as extensively characterized as its E. coli counterpart, researchers should initially compare activities under various conditions to determine optimal parameters for this specific protein.