Recombinant Photobacterium profundum Crossover junction endodeoxyribonuclease RuvC (ruvC)

What is RuvC and what role does it play in bacterial DNA repair?

RuvC is a crossover junction endodeoxyribonuclease that plays a crucial role in DNA repair and homologous recombination. It specifically cleaves Holliday junctions, which are formed during genetic recombination and link two double-stranded DNA molecules with a single-stranded crossover. The enzyme catalyzes the resolution of these structures by introducing nicks into DNA strands with the same polarity, leaving a 5' terminal phosphate and a 3' terminal hydroxyl group that can be ligated by DNA ligases .

The active form of RuvC is a dimer, which is mechanistically suited for its function as an endonuclease involved in swapping DNA strands at crossover junctions. In Escherichia coli, the RuvC protein structure is a 3-layer alpha-beta sandwich containing a 5-stranded beta-sheet sandwiched between 5 alpha-helices. It requires magnesium ion binding for its catalytic activity .

How does P. profundum RuvC differ from other bacterial RuvC proteins?

While the core enzymatic function of RuvC is conserved across bacterial species, P. profundum RuvC has evolved to function under the extreme conditions of the deep sea environment. P. profundum is a deep-sea Gammaproteobacterium with the ability to grow at temperatures from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa depending on the strain .

The strain SS9, from which recombinant RuvC is often derived, exhibits optimal growth at 15°C and 28 MPa, making it both a psychrophile and a piezophile . This environmental adaptation suggests potential structural modifications in its DNA repair enzymes, including RuvC, that allow them to maintain functionality under high hydrostatic pressure.

Transcriptomic analysis has shown that genes encoding DNA repair proteins, including ruvC, show enhanced expression at 10 MPa in some Alcanivorax strains (closely related marine bacteria), suggesting that DNA repair mechanisms may be particularly important for adaptation to increased pressure .

What expression systems are most effective for producing recombinant P. profundum RuvC?

Expression of recombinant P. profundum RuvC is typically performed in E. coli expression systems, similar to other recombinant proteins. Based on the available commercial recombinant P. profundum RuvC products, E. coli appears to be the preferred expression host . When expressing piezophilic proteins in mesophilic hosts like E. coli, several considerations should be taken into account:

Recommended Expression Protocol:

• Vector Selection: Use vectors with inducible promoters (such as T7 or tac) to control expression timing

• Host Strain Selection: BL21(DE3) or its derivatives are commonly used for recombinant protein expression

• Growth Conditions: Consider lower growth temperatures (15-20°C) to mimic the native temperature of P. profundum

• Induction Parameters: Use lower concentrations of inducers and longer induction times at reduced temperatures

What are the critical factors affecting the solubility and activity of recombinant P. profundum RuvC?

Several factors can affect the solubility and activity of recombinant P. profundum RuvC:

When purifying the protein, it's essential to maintain these conditions to preserve the native conformation and activity of the enzyme. The addition of stabilizing agents and protease inhibitors can also help maintain protein integrity during purification.

What in vitro assays can be used to measure P. profundum RuvC activity?

To assess the endonuclease activity of recombinant P. profundum RuvC, researchers can employ several in vitro assays:

• Holliday Junction Cleavage Assay:

  • Synthetic four-way junctions labeled with fluorescent dyes or radioisotopes

  • Reaction products analyzed by gel electrophoresis

  • Quantification of cleaved products relative to substrate

• Cruciform Structure Cleavage:

  • Similar to E. coli RuvC studies, P. profundum RuvC cleaves cruciform junctions formed by the extrusion of inverted repeat sequences from supercoiled plasmids

  • Analysis involves comparing nicked and linear forms after enzyme treatment

• Pressure-Modulated Activity Assays:

  • Custom high-pressure chambers can be used to assess activity under different hydrostatic pressures

  • This is particularly relevant for understanding the pressure adaptation of P. profundum RuvC

How can researchers measure the effects of pressure on RuvC activity?

Measuring enzyme activity under pressure requires specialized equipment and techniques:

Methodological Approach:

• High-Pressure Reaction Vessels:

  • Use stainless steel pressure vessels capable of maintaining stable pressure conditions

  • Include optical windows for real-time spectroscopic measurements if possible

• Pressure-Resistant Substrates:

  • Design fluorogenic substrates that can be measured through pressure-resistant windows

  • Monitor changes in fluorescence as a function of enzyme activity

• Comparative Analysis Protocol:

  • Test activity at various pressures (0.1 MPa, 5 MPa, 10 MPa, 28 MPa)

  • Compare with mesophilic RuvC proteins from non-piezophilic organisms as controls

  • Measure reaction rates and substrate affinity at each pressure point

• Post-Pressure Analysis:

  • After decompression, analyze reaction products by standard methods (gel electrophoresis)

  • Compare with reactions conducted at atmospheric pressure

Research has shown that in the related marine bacterium Alcanivorax dieselolei, genes encoding DNA repair proteins such as ruvC show enhanced expression at 10 MPa compared to atmospheric pressure (0.1 MPa), suggesting functional importance under pressure conditions .

How does the relationship between RuvC and RecG differ in P. profundum compared to other bacteria?

The relationship between RuvC and RecG in recombination pathways has been extensively studied in E. coli but less so in P. profundum. In E. coli, recombination is not highly dependent on RuvC alone, suggesting multiple pathways to produce recombinants. Lloyd found that recombination in a ruvC mutant depends on RecG, indicating complementary roles .

The interplay between these proteins may be particularly significant in P. profundum due to pressure adaptation:

• Comparative Function:

  • In E. coli, RecG helicase can process three-stranded junctions to generate recombinants without endonucleolytic resolution of Holliday junctions

  • RuvC provides junction-specific endonuclease activity expected of a Holliday junction resolvase

• Pressure Adaptations:

  • Research on P. profundum strain SS9 has shown that RecD function is essential for high-pressure growth

  • This suggests that DNA recombination and repair pathways, which include RuvC and RecG, may have unique adaptations for high-pressure environments

• Potential Mechanistic Differences:

  • The enhanced expression of ruvC under pressure conditions in marine bacteria suggests that RuvC may play a more critical role in recombination under pressure than at atmospheric pressure

  • This could indicate a shift in the balance between RuvC and RecG pathways as a function of pressure

How do pressure and temperature interact to affect P. profundum RuvC activity?

P. profundum SS9 is both a piezophile (pressure-loving) and a psychrophile (cold-loving), with optimal growth at 15°C and 28 MPa . This dual adaptation suggests complex interactions between temperature and pressure effects on its enzymes, including RuvC:

Temperature-Pressure Interaction Effects:

In the related marine bacterium Alcanivorax dieselolei, transcriptome analysis showed that under mild pressure (10 MPa), most genes were downregulated, but specific stress response genes were upregulated, including the DNA repair gene ruvC . This suggests that maintaining DNA repair capacity is particularly important for adaptation to increased pressure.

How can researchers create and verify ruvC mutants in P. profundum?

Creating and verifying ruvC mutants in P. profundum can be approached using similar techniques to those employed for creating recD mutants in P. profundum as described in the research literature :

Protocol for Creating ruvC Mutants:

• PCR Amplification of ruvC Fragment:

  • Design primers to amplify an internal portion of the ruvC gene

  • PCR conditions: 92°C for 1 min, 48°C for 1 min, and 72°C for 1 min for 25 cycles

• Cloning into Suicide Vector:

  • Clone the PCR product into an intermediate vector (e.g., pCR2.1)

  • Subclone into a suicide vector such as pMUT100

• Conjugal Transfer:

  • Conjugate the resulting plasmid into P. profundum wild-type strain

  • Perform matings at room temperature for 12-16 hours

  • After incubation, wash cells and plate onto selective medium

  • Incubate at 15°C for 3-5 days

• Verification of Integration:

  • Exconjugants arising from pMUT100-based plasmids result from the entire plasmid integrating into the genome in a single-crossover event

  • Verify integration by PCR and sequencing

  • Confirm disruption of the ruvC gene

What phenotypic changes can be expected in P. profundum ruvC mutants under varying pressure conditions?

Based on research with recD mutants in P. profundum and ruvC mutants in other bacteria, several phenotypic changes might be expected:

Predicted Phenotypic Effects of ruvC Mutation:

• Growth Defects Under Pressure:

  • Similar to recD mutants, ruvC mutants might show pressure-sensitive growth phenotypes

  • The magnitude of the defect may depend on the extent of disruption of the ruvC gene

• DNA Damage Sensitivity:

  • Increased sensitivity to DNA-damaging agents, especially those causing double-strand breaks

  • This sensitivity might be more pronounced under high pressure conditions

• Recombination Deficiency:

  • Reduced frequency of homologous recombination

  • This effect may be particularly evident in crosses with specific genetic markers

• Compensatory Mechanism Activation:

  • Potential upregulation of alternative recombination pathways, possibly involving RecG

  • This is based on the observation in E. coli that recombination in a ruvC mutant is dependent on RecG

• Cell Morphology Changes:

  • Possible filamentation or other morphological abnormalities under high pressure

  • This is suggested by the observation that RecD function affects cell morphology under pressure in E. coli

How does P. profundum RuvC compare functionally with RuvC from mesophilic bacteria?

A comparison between P. profundum RuvC and RuvC from mesophilic bacteria like E. coli reveals insights into pressure adaptation:

Comparative Analysis Table:

What insights can be gained by studying the evolution of RuvC in piezophilic bacteria?

Studying the evolution of RuvC in piezophilic bacteria like P. profundum provides insights into molecular adaptation to extreme environments:

• Evolutionary Adaptations:

  • Comparative sequence analysis may reveal specific amino acid substitutions that contribute to pressure tolerance

  • These often involve changes that affect protein flexibility, stability, and volume changes during catalysis

• Selection Pressures:

  • RuvC likely experienced strong selection pressure in deep-sea bacteria due to the importance of DNA repair in high-pressure environments

  • Transcriptomic evidence shows upregulation of DNA repair genes, including ruvC, under pressure conditions

• Convergent Evolution:

  • Determining whether similar adaptations have evolved independently in different piezophilic bacteria

  • This can identify critical residues and structural features necessary for pressure adaptation

• Environmental Correlation:

  • Correlation between RuvC sequence variations and the depth/pressure of isolation environments

  • Different P. profundum strains (SS9, 3TCK, DSJ4, 1230) isolated from different depths may show variations in their RuvC proteins that correlate with their native pressure environments

How can P. profundum RuvC be utilized in high-pressure biotechnology applications?

The pressure-adapted properties of P. profundum RuvC open several potential biotechnology applications:

• High-Pressure DNA Manipulation:

  • Development of DNA recombination systems that function under high pressure

  • This could be valuable for deep-sea bioprospecting and in situ genetic manipulation

• Pressure-Stable Molecular Tools:

  • Engineering of chimeric enzymes incorporating pressure-stable domains from P. profundum RuvC

  • Creation of pressure-resistant DNA modifying enzymes for specialized applications

• Structural Biology Studies:

  • Using P. profundum RuvC as a model to understand protein adaptation to extreme conditions

  • Insights gained could inform the design of other pressure-stable proteins

• Environmental DNA Repair:

  • Development of enzyme systems for DNA repair in high-pressure environments

  • Potential applications in bioremediation of deep-sea oil spills

What are the current challenges in studying protein-protein interactions involving RuvC under high pressure?

Studying protein-protein interactions under high pressure presents several technical challenges:

Technical Challenges and Potential Solutions:

Understanding these interactions is crucial because RuvC functions in concert with other recombination proteins. For example, in E. coli, the dependence of Red-mediated recombination on RuvC varied considerably among different types of crosses, and the frequency of recombination was higher in recG- than in recG+ strains , suggesting complex protein-protein interactions that may be further modified under pressure.

What are the most promising research directions for understanding P. profundum RuvC function?

Several promising research directions could advance our understanding of P. profundum RuvC:

• Structural Studies:

  • Determination of the three-dimensional structure of P. profundum RuvC under various pressure conditions

  • Identification of specific structural elements that contribute to pressure adaptation

• Functional Genomics:

  • Comprehensive analysis of the effects of ruvC mutations on global gene expression under different pressure conditions

  • Identification of genetic interactions and compensatory pathways

• Comparative Enzymology:

  • Detailed kinetic analysis comparing RuvC from P. profundum with homologs from non-piezophilic bacteria

  • Determination of pressure-dependent changes in catalytic parameters

• Ecological Relevance:

  • Investigation of the role of RuvC in P. profundum adaptation to natural deep-sea environments

  • Understanding how DNA repair mechanisms contribute to survival in extreme habitats

How might studying P. profundum RuvC contribute to our understanding of extremophile adaptation?

Research on P. profundum RuvC has broader implications for understanding extremophile adaptation:

• Molecular Basis of Piezophily:

  • RuvC represents an essential cellular function that must adapt to extreme pressure

  • Understanding these adaptations provides insights into general principles of protein evolution under pressure

• Convergent Evolution:

  • Comparison with other extremophile RuvC proteins can reveal whether similar solutions have evolved independently

  • This helps identify critical adaptations versus neutral variations

• Multiple Stress Adaptation:

  • P. profundum is both a piezophile and psychrophile , allowing study of adaptations to multiple stressors

  • The interplay between pressure and cold adaptation in RuvC can illuminate how proteins evolve to function under multiple extreme conditions

• Limits of Life:

  • Understanding how essential cellular processes like DNA repair function under extreme conditions helps define the boundaries of life

  • This has implications for astrobiology and the search for life in extreme environments