Recombinant Thermus thermophilus Holliday junction ATP-dependent DNA helicase RuvA (ruvA)

What is RuvA and what role does it play in DNA recombination?

RuvA is a Holliday junction-specific DNA-binding protein that, together with RuvB (a DNA helicase), promotes branch migration of Holliday junctions during homologous recombination . Holliday junctions are key intermediates formed during DNA recombination across all kingdoms of life, representing the point where two homologous DNA molecules exchange genetic information . In bacteria such as Thermus thermophilus and Escherichia coli, RuvA forms a complex with RuvB that targets the helicase activity of RuvB to the Holliday junction, facilitating the exchange of DNA strands . This process is essential for proper recombination and DNA repair, allowing organisms to maintain genetic integrity and generate genetic diversity.

How does the structure of T. thermophilus RuvA differ from other bacterial RuvA proteins?

The structure of T. thermophilus RuvA shares fundamental similarities with other bacterial RuvA proteins, such as those from E. coli, but also exhibits distinct features related to its thermostability. T. thermophilus RuvA has a more tightly associated dimer interface, likely contributing to its stability at high temperatures . When examining the crystal structure of T. thermophilus RuvC (which works in conjunction with RuvAB), researchers observed distinct asymmetry near the dimer interface in regions containing catalytically important aromatic residues . This structural asymmetry might be functionally significant for the thermostability and activity of the entire RuvABC complex in T. thermophilus. Additionally, time-resolved cryo-electron microscopy has revealed seven distinct conformational states of the T. thermophilus RuvAB complex during Holliday junction processing, offering insights into the unique structural dynamics of this thermophilic protein complex .

What are the most effective methods for expressing recombinant T. thermophilus RuvA?

Expressing recombinant T. thermophilus RuvA typically involves heterologous expression in E. coli expression systems, which has proven successful for numerous thermophilic proteins . For optimal expression, researchers should consider the following methodology:

• Gene cloning strategy: The ruvA gene can be PCR-amplified from T. thermophilus genomic DNA using specific primers containing appropriate restriction sites for subsequent cloning into expression vectors.

• Expression vector selection: Vectors containing strong promoters like T7 (pET series) are generally suitable for high-level expression. Including a His-tag facilitates subsequent purification.

• Expression conditions: Induction at lower temperatures (25-30°C) for longer periods often increases the solubility of recombinant thermophilic proteins in E. coli.

• Purification advantage: One significant benefit of expressing thermophilic proteins is the possibility of a heat treatment purification step. After cell lysis, heating the crude extract to 65-70°C precipitates most E. coli proteins while leaving the thermostable T. thermophilus RuvA in solution .

As demonstrated with other T. thermophilus proteins, this approach can yield high amounts (>30mg/L) of purified, active recombinant protein without requiring extensive chromatography steps .

How can zero-mode waveguides (ZMWs) be utilized to study RuvA-RuvB-Holliday junction complex formation?

Zero-mode waveguides (ZMWs) represent an advanced technique for studying RuvA-RuvB-Holliday junction complex formation at the single-molecule level . The methodology involves:

• Preparation of ZMWs: Nanosized holes (typically 50-200 nm in diameter) are fabricated in a thin metal film deposited on a glass substrate.

• Sample preparation: Fluorescently labeled components (typically Cy5-labeled RuvB) are used together with unlabeled RuvA and Holliday junction DNA.

• Observation procedure: The small observation volume of ZMWs (zeptoliters) enables visualization of single fluorescent molecules even at micromolar concentrations of labeled proteins.

• Data analysis: By counting the number of RuvB molecules binding to RuvA-Holliday junction complexes under different nucleotide conditions, researchers can quantitatively assess complex formation efficiency.

Studies using this technique have demonstrated that different nucleotide analogs increased the amount of Cy5-RuvB binding to RuvA-Holliday junction DNA complex in the following order: no nucleotide < ADP < ATPγS < mixture of ADP and ATPγS . This suggests that both ATP binding and ATP hydrolysis by RuvB contribute to stable complex formation, providing important mechanistic insights into the nucleotide-dependent assembly of the active RuvAB machinery .

How does ATP influence the RuvA-RuvB-Holliday junction complex formation and function?

ATP plays multiple critical roles in the formation and function of the RuvA-RuvB-Holliday junction complex, as revealed by several experimental approaches :

• Complex assembly: ATP binding to RuvB significantly enhances the formation of stable RuvA-RuvB-Holliday junction complexes. Experiments using ZMWs demonstrated that ATP analogs increased RuvB binding to RuvA-Holliday junction complexes, with a mixture of ADP and ATPγS showing the highest efficiency .

• Thermostability: T. thermophilus RuvB requires ATP for its thermostability, as judged by its ATPase activity at high temperatures .

• Mechanical force generation: Time-resolved cryo-electron microscopy studies have revealed that ATP hydrolysis enables RuvB to convert energy into a lever motion, which generates the pulling force that drives branch migration .

• Conformational changes: The complete nucleotide cycle involves coordinated motions in a "converter" formed by DNA-disengaged RuvB subunits, which stimulate hydrolysis and nucleotide exchange .

These findings suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates stable RuvA-RuvB-Holliday junction DNA complex formation and function .

What is the mechanism of ATP-dependent branch migration by the RuvAB complex?

The ATP-dependent branch migration mechanism of the RuvAB complex involves a sophisticated sequence of events revealed through time-resolved cryo-electron microscopy studies :

• Initial assembly: RuvA specifically binds to the Holliday junction, creating a platform for RuvB hexameric rings to assemble on the DNA arms.

• Nucleotide cycle: The RuvB hexamers undergo a complete nucleotide cycle involving:

  • ATP binding

  • Conformational changes

  • ATP hydrolysis

  • Nucleotide exchange

• Energy conversion: Coordinated motions in a "converter" region formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. When the converter becomes immobilized, RuvB converts ATP energy into a lever motion.

• Force generation: This lever motion generates the pulling force that drives strand exchange at the Holliday junction.

• Rotation mechanism: Importantly, the RuvB motors rotate together with the DNA substrate, which, combined with the progressing nucleotide cycle, forms the mechanistic basis for continuous branch migration .

This coordinated process allows for the ATP-dependent processing of Holliday junctions during homologous recombination, facilitating proper genome maintenance and genetic exchange.

What unique properties does T. thermophilus RuvB exhibit compared to mesophilic homologs?

T. thermophilus RuvB exhibits several distinctive properties compared to its mesophilic counterparts, particularly E. coli RuvB :

• Broader nucleotide utilization: T. thermophilus RuvB hydrolyzes a wider range of nucleoside triphosphates than E. coli RuvB, including GTP and dGTP, although only ATP hydrolysis supports branch migration.

• DNA substrate preference: E. coli RuvB shows strong ATPase stimulation by supercoiled DNA but only weak enhancement by linear duplex DNA. In contrast, T. thermophilus RuvB's ATPase activity is efficiently and equally enhanced by both supercoiled and linear duplex DNA .

• RuvA-independent activity: At elevated temperatures (60°C), T. thermophilus RuvB can promote branch migration of synthetic Holliday junctions in an ATP-dependent manner even in the absence of RuvA protein, an activity not observed with E. coli RuvB .

• Thermostability mechanism: T. thermophilus RuvB requires ATP for thermostability, as measured by its ATPase activity, indicating a unique structural dependency on nucleotide binding .

• Cross-species compatibility: Despite its unique properties, T. thermophilus RuvB can functionally interact with E. coli RuvA, which greatly enhances its ATP hydrolysis and branch migration activities at lower temperatures (37°C) .

These unusual properties likely reflect adaptations to the high-temperature environment of T. thermophilus and may provide insights into protein engineering for enhanced thermostability.

How does temperature affect the activity and stability of recombinant T. thermophilus RuvA?

The activity and stability of recombinant T. thermophilus RuvA, like other proteins from this thermophilic organism, are significantly influenced by temperature in several ways:

• Optimal activity temperature: While T. thermophilus grows optimally around 70°C, many of its enzymes show peak activity at even higher temperatures. For the RuvAB complex, efficient branch migration has been observed at temperatures up to 60°C in vitro .

• Thermal stability: T. thermophilus proteins typically demonstrate remarkable thermostability, maintaining structural integrity and function at temperatures that would denature mesophilic proteins. This stability is often attributed to:

  • Increased hydrophobic interactions

  • Higher number of salt bridges

  • More compact packing of amino acid residues

  • Reduced flexibility at room temperature

• Temperature-dependent complex formation: Studies have shown that elevated temperatures (50-60°C) are required for optimal RuvB-mediated branch migration in the absence of RuvA, whereas at lower temperatures (37°C), RuvA significantly enhances RuvB activity .

• Storage considerations: Purified recombinant T. thermophilus RuvA can generally be stored for extended periods at 4°C or even room temperature without significant loss of activity, unlike mesophilic proteins that typically require storage at -20°C or -80°C.

These temperature-dependent properties make T. thermophilus RuvA an excellent model for studying thermostable protein structure-function relationships and a valuable tool for high-temperature biochemical applications.

How do genetic studies of ruvA and related genes in T. thermophilus inform our understanding of DNA repair pathways?

Genetic studies of ruvA and related genes in T. thermophilus have provided valuable insights into DNA repair pathways in thermophilic bacteria and their evolutionary conservation :

• Differential requirements: Knockout studies in T. thermophilus have revealed that components of DNA repair pathways, such as the RecFOR pathway which works upstream of RuvAB, have differential roles rather than functioning as a single unit. This contrasts with their functions in other bacteria .

• Epistatic relationships: Genetic analyses have uncovered an epistatic relationship between the AddAB complex (a helicase-exonuclease important for recombination) and the RecFOR pathway in T. thermophilus, suggesting coordinated activities in DNA repair .

• Conservation of interactions: Despite the unique properties of T. thermophilus RuvB, its ability to interact functionally with E. coli RuvA indicates conservation of critical protein-protein interfaces across different bacterial species. This suggests the existence of a ruvA homolog in T. thermophilus before it was explicitly identified .

• Viability impacts: Deletion of ruvB in T. thermophilus leads to DNA repair defects and increased filamentation phenotypes, indicating its importance in chromosome segregation and maintenance of genomic integrity .

• Cross-complementation limitations: Interestingly, the T. thermophilus ruvB gene could not complement the UV sensitivity of an E. coli ruvB deletion mutant and actually made wild-type E. coli strains more sensitive to UV, suggesting incompatibilities in the complete repair pathway despite conserved protein interactions .

These genetic findings highlight the adaptability of DNA repair systems across different bacterial species while maintaining core mechanistic features.

What roles do RuvA and RuvB play in the natural habitat of T. thermophilus?

In the natural habitat of T. thermophilus, which includes terrestrial hot springs with temperatures above 60°C and neutral pH , RuvA and RuvB play essential roles in genome maintenance and adaptation:

• DNA damage repair: High temperatures accelerate DNA damage processes, including depurination and deamination. The RuvAB complex, along with RuvC, provides crucial DNA repair capacity through homologous recombination .

• Horizontal gene transfer: T. thermophilus is naturally competent and can acquire DNA from its environment. Genes encoding metabolic capabilities like nitrate and nitrite respiration are easily transferred between strains by natural competence or conjugation-like processes . The RuvAB system likely plays a role in integrating this foreign DNA through recombination events.

• Phage defense: Hot springs harbor diverse phages that infect Thermus species . The CRISPR-Cas systems and other immunity mechanisms in T. thermophilus suggest ongoing interactions with these viruses. The RuvAB system might contribute to DNA repair following phage infection or participate in recombination events that affect CRISPR adaptation.

• Genome flexibility: The RuvAB complex contributes to the genomic plasticity that allows T. thermophilus to adapt to changing environmental conditions. This flexibility is reflected in the extended "pangenome" of T. thermophilus, which includes plasmid-associated genetic islands that provide enhanced adaptability .

• Stress response: Under stressful conditions, including temperature fluctuations in hot springs, increased DNA damage would necessitate heightened activity of repair enzymes like the RuvAB complex.

The adaptation of RuvA and RuvB to function efficiently at high temperatures is therefore critical to the survival and evolution of T. thermophilus in its extreme habitat.

How can structural insights from T. thermophilus RuvAB studies contribute to understanding other AAA+ ATPase systems?

Structural studies of T. thermophilus RuvAB provide valuable insights into the fundamental mechanisms of AAA+ ATPases, with broad implications for understanding related systems :

• Nucleotide cycle mapping: Time-resolved cryo-electron microscopy of the ATP-hydrolysing RuvAB complex has revealed seven distinct conformational states, capturing the complete nucleotide cycle. This comprehensive view of sequential transition-state intermediates serves as a blueprint for understanding the chemo-mechanical coupling in other hexameric AAA+ motors .

• Energy conversion mechanism: The identification of a "converter" mechanism in RuvB, where coordinated motions in DNA-disengaged subunits stimulate hydrolysis and nucleotide exchange, provides a model for how other AAA+ ATPases might convert chemical energy into mechanical force .

• Substrate interaction dynamics: The discovery that RuvB motors rotate together with the DNA substrate during branch migration reveals principles of how AAA+ motors can maintain continuous engagement with their substrates while undergoing conformational changes .

• Subunit coordination: The spatiotemporal relationship between ATP hydrolysis, nucleotide exchange, and context-specific conformational changes in RuvB illustrates how adjacent subunits in hexameric rings can coordinate their activities, a principle likely applicable to other ring-shaped molecular machines .

• Drug design opportunities: The elucidation of discrete and sequential transition states provides a blueprint for the design of state-specific compounds targeting AAA+ motors, which could have therapeutic applications for human AAA+ ATPases involved in disease processes .

These insights from T. thermophilus RuvAB can inform research on other AAA+ systems involved in protein degradation, membrane fusion, DNA replication, and various other cellular processes.

What are the potential applications of recombinant T. thermophilus RuvA in enhancing PCR and other molecular biology techniques?

Recombinant T. thermophilus RuvA has several potential applications in enhancing molecular biology techniques, drawing on its thermostability and specialized interactions with DNA:

• PCR enhancement: Similar to how T. thermophilus RecA has been shown to enhance PCR signals of DNA viruses like Hepatitis B virus , RuvA could potentially improve amplification of certain DNA templates by:

  • Facilitating strand separation at difficult secondary structures

  • Stabilizing primer-template complexes at high temperatures

  • Enhancing processivity of DNA polymerase through protein-protein interactions

• Holliday junction manipulation: Purified RuvA could be used in vitro to stabilize and manipulate Holliday junctions for various applications:

  • Structural studies of DNA recombination intermediates

  • Creation of stable four-way junctions for nanotechnology applications

  • Template preparation for studying other junction-processing enzymes

• Thermostable complex formation: The ability of T. thermophilus RuvA to form stable complexes with DNA at high temperatures could be exploited in techniques requiring elevated temperatures:

  • Isothermal amplification methods

  • High-temperature DNA shuffling procedures

  • Thermophilic in vitro recombination systems

• Diagnostic applications: The specificity of RuvA for Holliday junctions could be harnessed in diagnostic methods:

  • Detection of recombination intermediates in cell-free systems

  • Development of structure-specific DNA detection platforms

  • Enhancement of sensitivity in nucleic acid detection assays

These applications leverage the unique properties of T. thermophilus RuvA, including its thermostability, specific DNA binding characteristics, and interaction capabilities with other recombination proteins.

How do the biochemical properties of T. thermophilus RuvA and RuvB compare with their counterparts in mesophilic bacteria?

The biochemical properties of T. thermophilus RuvA and RuvB differ significantly from their mesophilic counterparts, particularly those from E. coli, in several key aspects :

These differences highlight how evolutionary adaptations to extreme environments can modify the biochemical properties of functionally conserved proteins, providing insights into structure-function relationships and protein engineering strategies .

What insights have crystallographic and cryo-EM studies provided about the structure-function relationship of T. thermophilus RuvA?

Crystallographic and cryo-electron microscopy studies have provided profound insights into the structure-function relationship of T. thermophilus RuvA and its interaction with RuvB and Holliday junction DNA :

These structural insights provide a molecular framework for understanding how the RuvAB complex performs its essential function in homologous recombination and DNA repair, with implications for protein engineering and drug design targeting AAA+ ATPases .

What are the most promising future research directions for T. thermophilus RuvA studies?

Several promising research directions for T. thermophilus RuvA studies could significantly advance our understanding of DNA recombination mechanisms and expand biotechnological applications:

• Single-molecule dynamics: Applying advanced single-molecule techniques to monitor real-time conformational changes and DNA movement by the RuvAB complex could reveal transient intermediates and kinetic parameters of branch migration .

• Interaction with other recombination proteins: Investigating how T. thermophilus RuvA and RuvB interact with other recombination proteins like RecA, RecG, and RuvC could provide a more comprehensive understanding of the recombination machinery in thermophiles .

• Protein engineering: Using insights from T. thermophilus RuvA structure to engineer mesophilic RuvA proteins with enhanced thermostability could create valuable tools for biotechnological applications requiring high-temperature DNA processing.

• Comparative genomics and evolution: Expanded analysis of RuvA variants across different thermophilic species could illuminate the evolutionary adaptations that enable protein function at high temperatures and the co-evolution of interacting partners.

• In vivo dynamics: Developing methods to track RuvA localization and activity in living T. thermophilus cells would provide insights into the spatial and temporal regulation of recombination in response to DNA damage.

• Application in synthetic biology: Exploring the potential of thermostable RuvA and RuvB as components in synthetic genetic circuits or DNA-based nanomachines that can operate at high temperatures.

• Cross-species functionality: Further investigation of the compatibility between recombination components from different species could reveal fundamental principles of protein-protein recognition and functional conservation.

These research directions would not only enhance our basic understanding of DNA recombination processes but could also lead to novel biotechnological applications of thermostable recombination proteins.

How might structural insights from T. thermophilus RuvA inform drug design targeting recombination proteins in pathogens?

Structural insights from T. thermophilus RuvA could inform drug design strategies targeting recombination proteins in pathogens through several avenues:

• Conserved active sites: Despite adaptations for thermostability, the core functional domains of RuvA are conserved across bacterial species. Detailed structural information from T. thermophilus RuvA could reveal druggable pockets present in pathogenic homologs .

• Nucleotide binding sites: The elucidation of different states in the ATP hydrolysis cycle of the RuvAB complex provides templates for designing nucleotide analogs that could selectively inhibit RuvB function in pathogens .

• Protein-protein interfaces: Structural studies revealing the RuvA-RuvB interface could guide the development of molecules that disrupt this essential interaction in pathogenic bacteria, potentially creating a new class of antibiotics .

• DNA binding surfaces: The detailed understanding of how RuvA recognizes and binds to Holliday junctions could enable the design of compounds that compete for this interaction, thereby inhibiting recombinational repair in pathogens .

• Transition state targeting: The identification of discrete and sequential transition-state intermediates in the RuvAB complex provides opportunities for designing inhibitors that trap the complex in non-productive conformations .

• Allosteric regulation sites: Structural studies might reveal allosteric sites that, when occupied by small molecules, could prevent the conformational changes necessary for RuvA function.

• Species-specific features: Comparative analysis of T. thermophilus RuvA with pathogenic homologs could identify structural differences that could be exploited for selective targeting, minimizing effects on the human microbiome.