Recombinant Rhodopirellula baltica Holliday junction ATP-dependent DNA helicase RuvB (ruvB)

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

RuvB is an ATP-dependent DNA helicase that functions as a molecular motor in homologous recombination, a fundamental process for maintaining genetic integrity across all domains of life . The protein assembles as a hexameric ring around double-stranded DNA and, in concert with RuvA, facilitates the branch migration of Holliday junctions, which are key intermediates formed during DNA recombination . By utilizing energy from ATP hydrolysis, RuvB generates the mechanical force necessary to translocate Holliday junctions along DNA, progressively exchanging one DNA strand for another in a process termed branch migration . This activity determines the amount of genetic information transferred between recombining partners and is essential for homologous recombination and recombinational repair . In bacteria like Rhodopirellula baltica, RuvB works within a coordinated machinery that includes RuvA, which recognizes and binds to Holliday junctions, and often RuvC, which ultimately resolves these structures into duplex products .

What is the structural organization of the RuvAB-Holliday junction complex?

The RuvAB-Holliday junction complex represents a sophisticated molecular machine with distinct architectural components that work together to facilitate branch migration. RuvA assembles as a tetramer that binds to the Holliday junction with high affinity and unfolds it from a stacked X-structure into a square-planar conformation, which optimizes it for branch migration . Two hexameric rings of RuvB encircle opposing DNA duplex arms of the junction and function as ATP-dependent DNA motors that extrude heteroduplex DNA . Recent cryo-EM studies have revealed that RuvB assembles into a spiral staircase-like hexamer around double-stranded DNA, with four protomers directly contacting the DNA backbone and a translocation step size of 2 nucleotides per ATP hydrolysis cycle . The interaction between RuvA and RuvB exhibits a 4:6 stoichiometry, reflecting an asymmetric engagement that supports the functional mechanics of branch migration . This structural arrangement enables the coordinated movement required for efficient branch migration, where the RuvB motors rotate together with the DNA substrate as part of the mechanistic basis for DNA recombination .

How does ATP hydrolysis by RuvB drive the branch migration process?

RuvB utilizes a sophisticated ATP hydrolysis mechanism to convert chemical energy into mechanical force for DNA translocation during branch migration. Each RuvB hexamer contains six ATP-binding sites, but the nucleotide states vary among subunits, supporting a sequential model for ATP hydrolysis and nucleotide recycling that occur at separate, singular positions within the ring . Time-resolved cryo-EM studies have captured the RuvAB complex in seven distinct conformational states that together reveal the complete nucleotide cycle and the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange, and context-specific conformational changes in RuvB . Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange, while immobilization of this converter enables RuvB to convert ATP energy into a lever motion that generates the pulling force driving branch migration . This process results in RuvB motors rotating together with the DNA substrate, which creates the mechanical basis for continuous branch migration of the Holliday junction . The energy conversion mechanism is highly efficient, with RuvB demonstrating processivity that can exceed the total length of experimental Holliday junction substrates under optimal conditions .

How does the RuvAB complex navigate through sequence heterologies during branch migration?

Branch migration must overcome sequence heterologies when recombination occurs between similar but not identical DNA molecules, which presents a significant challenge for the RuvAB machinery. Biochemical studies indicate that RuvAB can bypass long tracks of heterology, although with reduced efficiency compared to homologous sequences . When the RuvAB complex encounters sequence heterology, it may either stall temporarily at the heterologous region or proceed through it, generating mismatches, insertions, or deletions in the heteroduplex DNA products . Computer simulations have been employed to model this process as a random Poisson process with characteristic times for different stages, including the time of bypass through sequence heterology (τhet) and the lifetime of the complex stalled at a sequence heterology (τlife) . These simulations help researchers estimate parameters such as translocation rates and processivity under different conditions . The ability to bypass heterology is crucial for the biological function of RuvAB in processing recombination intermediates in vivo, where perfect sequence identity between recombining molecules is not always present.

What methodological approaches can reveal the conformational dynamics of RuvB during the ATP hydrolysis cycle?

Understanding the conformational dynamics of RuvB during ATP hydrolysis requires sophisticated structural biology techniques that can capture transitional states. Time-resolved cryo-EM has emerged as a powerful tool for this purpose, enabling researchers to visualize the RuvAB complex in multiple distinct conformational states corresponding to different stages of the ATP hydrolysis cycle . This approach involves rapid freezing of samples at various time points after initiating the reaction, followed by detailed image analysis to identify and classify particles representing different conformational states . 3D variability analysis in software packages like cryoSPARC can help identify the principal components of structural variability, while focused classification without alignment in Relion can isolate specific conformational subsets . To improve resolution for specific regions of interest, techniques such as focused refinement with soft masks, Bayesian polishing, and local refinement can be applied . Complementary approaches include single-molecule FRET to monitor conformational changes in real-time and hydrogen-deuterium exchange mass spectrometry to identify regions undergoing structural changes during the catalytic cycle.

How does the nucleotide state of individual RuvB subunits correlate with their position and function within the hexamer?

The RuvB hexamer displays an asymmetric organization where nucleotide states vary systematically around the ring, creating a coordinated system for DNA translocation. Structural studies have shown that the variation of nucleotide-binding states in RuvB supports a sequential model for ATP hydrolysis and nucleotide recycling, which occur at separate, singular positions within the hexamer . ATP hydrolysis most likely occurs at the top position of the spiral staircase in a sequential manner, with each hydrolysis event triggering conformational changes that propagate around the ring . This arrangement creates a "hand-over-hand" mechanism where subunits change conformation and position relative to the DNA substrate as they progress through the ATP hydrolysis cycle . The asymmetric assembly of RuvB also explains the 6:4 stoichiometry between the RuvB/RuvA complex and indicates a potential asymmetric engagement with RuvA during HJ branch migration . This intricate correlation between nucleotide state and subunit position/function ensures that energy from ATP hydrolysis is efficiently converted into the mechanical force needed for DNA translocation, with a step size of approximately 2 nucleotides per ATP hydrolysis cycle .

What expression systems and purification strategies are optimal for recombinant Rhodopirellula baltica RuvB?

Obtaining pure, properly folded recombinant R. baltica RuvB requires careful selection of expression systems and purification strategies tailored to this specific protein. Based on proteome analysis studies of R. baltica, which have successfully identified and characterized numerous proteins including those involved in DNA metabolism, a heterologous expression system using E. coli would be appropriate . The ruvB gene from R. baltica should be cloned into an expression vector containing an inducible promoter (such as T7) and a suitable affinity tag (6xHis or GST) to facilitate purification . Expression conditions need optimization, with particular attention to temperature (often lowered to 16-18°C) and induction parameters to maximize protein solubility rather than inclusion body formation. A multi-step purification protocol typically works best, beginning with affinity chromatography (Ni-NTA for His-tagged constructs), followed by ion exchange chromatography to separate differently charged species, and finally size exclusion chromatography to isolate properly assembled hexameric complexes . Buffer conditions require careful optimization, often including stabilizing agents such as glycerol (10-15%), reducing agents to maintain cysteine residues (DTT or β-mercaptoethanol), and appropriate salt concentrations to maintain solubility while permitting proper oligomerization .

What experimental approaches can assess the ATP-dependent branch migration activity of recombinant RuvB?

Multiple complementary approaches can be employed to rigorously characterize the ATP-dependent branch migration activity of recombinant RuvB. Gel-based branch migration assays utilize synthetic Holliday junction substrates, often with differentially labeled DNA strands, to monitor the conversion of junctions to product DNA molecules over time by gel electrophoresis . In these assays, the position of the crossover point in the Holliday junction can create multiple bands (labeled as HJ* in experimental results), and restriction enzyme mapping can be used to properly identify the molecular species generated during branch migration . To obtain kinetic parameters, time-course experiments can be performed, with samples taken at various time points and analyzed to determine the rate of product formation . Computer simulations can help interpret these results by separating the kinetics of branch migration into individual reaction steps, estimating parameters such as translocation rate (e.g., 9±0.1 bp/s at 20°C) and processivity . More sophisticated techniques include single-molecule approaches using fluorescently labeled DNA substrates and FRET (Förster Resonance Energy Transfer) to monitor branch migration in real-time, providing insights into individual molecular events rather than bulk behavior .

How can structural studies best elucidate the mechanism of RuvB-mediated branch migration?

Structural studies of RuvB require integrated approaches that capture both static structures and dynamic conformational changes during the branch migration process. Cryo-electron microscopy (cryo-EM) has emerged as the method of choice for studying RuvB-DNA complexes, capable of yielding high-resolution structures as demonstrated by recent studies achieving resolutions of 3.01-7.02 Å for different regions of the RuvAB-Holliday junction complex . The workflow typically involves sample preparation with careful optimization of protein:DNA ratios, vitrification conditions, image acquisition with motion correction, particle selection, 2D and 3D classification, and refinement using software packages such as Relion 3.1 and cryoSPARC . For addressing the inherent conformational heterogeneity of RuvB during its catalytic cycle, techniques such as 3D focused classification without alignment and 3D variability analysis can identify and sort different conformational states . To improve resolution in specific regions, techniques such as focused refinement with soft masks, Bayesian polishing, and homogeneous refinement can be applied . Complementary approaches include hydrogen-deuterium exchange mass spectrometry to identify regions undergoing conformational changes and molecular dynamics simulations to model transitions between observed states and predict functional motions not captured in static structures .

How should researchers interpret variability in measured RuvB translocation rates across different studies?

Variability in RuvB translocation rates reported across different studies requires careful consideration of experimental conditions and analytical approaches. Factors influencing translocation rates include temperature (with significant differences observed between measurements at 20°C versus 37°C), buffer composition (particularly divalent cation concentrations), DNA substrate structure and sequence, and the presence of accessory proteins like RuvA . When faced with apparently contradictory results, researchers should consider using computer simulations to separate the multistage process of branch migration into individual reaction steps, as demonstrated in studies that modeled branch migration as a series of random Poisson processes with characteristic times for different stages . This approach can help determine whether observed differences result from different rate-limiting steps dominating under varying experimental conditions . Additionally, researchers should distinguish between bulk measurements, which average across a population of molecules, and single-molecule techniques, which can reveal heterogeneity in behavior . Creating comprehensive models that incorporate multiple parameters (velocity, processivity, assembly time, heterology bypass efficiency) rather than focusing solely on translocation rate can help reconcile apparently contradictory observations across different experimental systems .

What approaches can resolve the structural heterogeneity inherent in RuvB-DNA complexes during cryo-EM analysis?

Resolving structural heterogeneity in RuvB-DNA complexes during cryo-EM analysis requires sophisticated computational approaches and strategic experimental design. The inherent conformational dynamics of RuvB during its ATP hydrolysis cycle creates significant challenges for structural determination, as demonstrated in studies where initial reconstructions yielded maps with varied resolution across different regions of the complex . To address this heterogeneity, researchers have successfully employed 3D focused classification without alignment in Relion combined with 3D variability analysis in cryoSPARC to identify particle subsets with improved density in regions of interest . After identifying these subsets, duplicate particles can be removed, and the selected particles subjected to heterogeneous refinement followed by homogeneous refinement with appropriate soft masks . For regions with particularly high variability, local refinement with specially designed masks and map sharpening with negative B factors can significantly improve local resolution . These approaches have enabled researchers to obtain detailed structures of different regions of the RuvAB-HJ complex, with resolutions ranging from 3.01 Å for the more stable RuvA region to 7.02 Å for the more dynamic RuvB region . Complementary approaches include using nucleotide analogs or transition state mimics to trap specific conformational states and applying time-resolved cryo-EM to capture the complex at different stages of its catalytic cycle .

How do in vitro measurements of RuvB activity translate to in vivo recombination processes?

Translating in vitro measurements of RuvB activity to in vivo recombination processes requires careful consideration of the cellular environment and physiological context. In vitro studies, which have measured parameters such as branch migration rates (~9 bp/s at 20°C) and processivity, provide valuable quantitative data but may differ from in vivo rates due to several factors . The cellular environment includes molecular crowding, which can alter protein-DNA interactions and reaction kinetics, as well as different ion concentrations and pH conditions compared to optimized buffer systems used in vitro . In vivo, RuvB operates as part of a larger recombination machinery that includes not only RuvA and RuvC but potentially other recombination and repair proteins that may influence its activity . Additionally, the ability of RuvAB to bypass sequence heterologies, although with reduced efficiency, has important implications for recombination between similar but not identical DNA molecules in bacterial genomes . Temperature is another critical factor, as demonstrated by significant differences in activity between 20°C and 37°C in vitro, suggesting that organisms living at different temperatures may experience different recombination kinetics . Researchers should therefore be cautious about directly extrapolating in vitro measurements to cellular processes without considering these contextual factors.

What are the most effective approaches for studying the energetics of RuvB-mediated branch migration?

What emerging technologies could advance our understanding of RuvB-mediated branch migration?

Several cutting-edge technologies hold promise for deepening our understanding of RuvB-mediated branch migration mechanisms and dynamics. Time-resolved cryo-EM with millisecond time resolution could capture additional transient states during the ATP hydrolysis cycle, revealing conformational intermediates that have thus far eluded detection . Integrating cryo-electron tomography with subtomogram averaging could visualize RuvAB complexes in cellular contexts, providing insights into how these machines function within the crowded environment of the cell . Advanced single-molecule techniques, including combined fluorescence and force spectroscopy, could simultaneously monitor conformational changes in RuvB and the mechanical forces generated during branch migration, directly connecting structural dynamics to function . Computational approaches such as molecular dynamics simulations that incorporate information from cryo-EM structures could model the complete conformational cycle of RuvB with atomistic detail, predicting energy barriers and identifying key residues involved in energy transduction . CRISPR-based genome editing approaches could enable precise manipulation of RuvB in model organisms, allowing researchers to test structure-function hypotheses in vivo and assess the physiological consequences of specific mutations . These technological advances, particularly when used in combination, have the potential to resolve outstanding questions about the detailed mechanics of branch migration and the coordination between RuvA and RuvB during this process.

How might comparative studies of RuvB from diverse organisms, including Rhodopirellula baltica, inform our understanding of recombination mechanisms?

Comparative studies of RuvB proteins from diverse organisms can provide valuable evolutionary insights and reveal both conserved mechanisms and adaptive variations in recombination processes. Rhodopirellula baltica, a marine aerobic heterotrophic bacterium belonging to the Planctomycetes phylum, represents an interesting subject for such comparative studies due to its ecological niche and evolutionary position . Proteome analysis of R. baltica has successfully identified numerous proteins, though information specifically about its RuvB remains limited in the current literature . Comparing the sequence, structure, and function of RuvB across diverse bacterial phyla could identify conserved catalytic residues essential for ATP hydrolysis and DNA binding, as well as variable regions that might relate to adaptation to different environmental conditions such as temperature, salinity, or pH . Examining how RuvB interfaces with other recombination proteins across different bacterial lineages might reveal alternative regulatory mechanisms or functional partnerships . Additionally, studying RuvB from organisms with unusual genomic features, such as high GC content or frequent genomic islands, could provide insights into how the recombination machinery adapts to different genomic landscapes . Such comparative approaches could ultimately contribute to a more comprehensive understanding of the evolution and adaptation of DNA recombination mechanisms across diverse bacterial lineages.