Recombinant Nitrosomonas europaea Putative Holliday junction resolvase (NE1667)

What is NE1667 and what is its role in Nitrosomonas europaea?

NE1667 is a putative Holliday junction resolvase from Nitrosomonas europaea, a gram-negative obligate chemolithoautotroph that derives its energy and reducing power from the oxidation of ammonia to nitrite . Holliday junction resolvases (HJRs) are key enzymes in DNA recombination that resolve four-way DNA structures formed during homologous recombination processes . In Nitrosomonas europaea, this enzyme likely plays a critical role in maintaining genome integrity by processing Holliday junctions that form during DNA repair mechanisms.

The protein has been identified through genomic analysis of Nitrosomonas europaea (ATCC 19718), which has a single circular chromosome of 2,812,094 bp containing approximately 2,460 protein-encoding genes . As an ammonia-oxidizing bacterium participating in the biogeochemical nitrogen cycle, Nitrosomonas europaea requires efficient DNA repair mechanisms to maintain genomic stability in various environmental conditions.

What are the basic characteristics of recombinant NE1667?

The recombinant form of NE1667 is available for research purposes with the product code CSB-YP767698NHH . Its UniProt accession number is Q82U42 . As a recombinant protein, it is produced through expression of the NE1667 gene in a heterologous system, which allows for purification and subsequent characterization of the protein's biochemical properties.

The stability and activity of the recombinant protein depends on proper storage conditions. The shelf life of liquid formulations is typically 6 months at -20°C/-80°C, while lyophilized forms can maintain stability for up to 12 months at the same temperature range . This extended shelf life for the lyophilized form is due to reduced susceptibility to degradation in the absence of water.

How do Holliday junction resolvases function in DNA repair?

Holliday junction resolvases function by recognizing and cleaving four-way DNA structures (Holliday junctions) that form during homologous recombination. Homologous recombination is one of the major mechanisms by which cells repair double-strand breaks in DNA caused by factors such as ultraviolet radiation and harmful chemicals .

During homologous recombination, the broken DNA interacts with an undamaged copy to form a Holliday junction. The intact DNA strands serve as templates for repair of the broken strands. After repair, the Holliday junction must be resolved to allow proper DNA segregation and maintain genomic integrity . Holliday junction resolvases catalyze this resolution by introducing precise symmetrical cuts in the DNA at the junction, acting as molecular scissors. The cut strands are then rejoined to fully restore the DNA structure .

What structural families do Holliday junction resolvases belong to?

Based on detailed computer analysis of structural and evolutionary relationships, Holliday junction resolvases have evolved independently from at least four distinct structural folds:

• RNase H fold

• Endonuclease fold

• Endonuclease VII-colicin E fold

• RusA fold

Among these, the endonuclease fold encompasses the greatest diversity of nucleases, including archaeal HJRs, repair nucleases like RecB and Vsr, restriction enzymes, and various predicted nucleases with yet undetermined specific activities . Within the RNase H fold, there exists both the previously characterized RuvC family and a newly discovered family of predicted HJRs that is nearly ubiquitous in bacteria .

What experimental approaches are recommended for studying NE1667 activity?

To study the enzymatic activity of NE1667, researchers should consider employing the cruciform plasmid cleavage assay, which is particularly useful for evaluating the nicking function of Holliday junction resolvases. This method uses plasmids containing an inverted-repeat sequence that extrudes a cruciform structure when supercoiled . The assay can distinguish between coordinated dual incision (by a dimeric resolvase) and uncoordinated cleavage (by monomeric enzymes) based on the migration patterns of reaction products during gel electrophoresis.

Experimental procedure:

• Prepare a plasmid containing an inverted-repeat sequence (similar to pIRbke8mut used for GEN1 studies)

• Ensure the plasmid is supercoiled to promote cruciform extrusion

• Incubate the plasmid with purified NE1667 under appropriate reaction conditions

• Analyze the reaction products by agarose gel electrophoresis

• Interpret results based on migration patterns:

  • Linear duplex products (slow migration): indicates coordinated dual incision

  • Nicked plasmids (even slower migration): indicates uncoordinated cleavage

This method can be supplemented with structure-guided mutations to validate mechanistic findings, similar to the approach used for human GEN1 .

How might the structure of NE1667 compare to other characterized Holliday junction resolvases?

While the specific structure of NE1667 has not been fully characterized in the provided search results, we can make informed predictions based on known structures of other Holliday junction resolvases.

The human Holliday junction resolvase GEN1 belongs to the Rad2/XPG nuclease family and contains a characteristic nuclease core supplemented with a chromodomain as an additional DNA interaction site . The chromodomain directly contacts DNA and its truncation severely hampers catalytic activity . This domain arrangement allows GEN1 to recognize Holliday junctions specifically among various DNA structures.

For bacterial Holliday junction resolvases, structural diversity exists across different families. The RuvC family is well-characterized within the RNase H fold, while additional families remain to be fully explored . The structural features that enable specific recognition of Holliday junctions versus other DNA structures are of particular interest for understanding substrate specificity.

A potential structural comparison of NE1667 with other resolvases might include:

Further structural studies using X-ray crystallography or cryo-electron microscopy would be valuable for elucidating the three-dimensional structure of NE1667 and its mode of interaction with DNA substrates.

How does the mechanism of Holliday junction resolution differ among various resolvase families?

Classical Holliday junction resolvases introduce two symmetrical incisions across the junction point by coordinating the action of two active sites. The first nick is rate-limiting, while the second one occurs near-simultaneously within the lifetime of the resolvase-DNA complex . This mechanism has been well-studied for bacterial and bacteriophage HJ resolvases.

For enzymes like GEN1, dimerization upon binding to HJ substrates is indicated by coordinated cleavage and by an increase in hydrodynamic radius compared to the protein alone . This suggests that proper dimer formation is crucial for the coordinated dual incision of Holliday junctions.

The mechanism of NE1667 specifically would need to be determined through biochemical characterization. Key aspects to investigate would include:

• Whether NE1667 functions as a monomer or dimer

• The coordination of nicking events during junction resolution

• The structural basis for substrate recognition

• The role of metal ions in catalysis

• The effect of DNA sequence context on resolution efficiency

Understanding these aspects of NE1667's mechanism would provide valuable insights into its role in the DNA repair pathway of Nitrosomonas europaea.

What is the potential relationship between NE1667 function and the unique metabolism of Nitrosomonas europaea?

Nitrosomonas europaea has a unique chemolithoautotrophic metabolism, deriving all its energy and reductant for growth from the oxidation of ammonia to nitrite . This specialized metabolism may create specific challenges for DNA integrity due to:

• Production of reactive nitrogen species during ammonia oxidation

• Oxidative stress from electron transport processes

• Potential DNA damage from environmental factors in its ecological niche

As a gram-negative obligate chemolithoautotroph participating in the biogeochemical nitrogen cycle, Nitrosomonas europaea likely requires robust DNA repair mechanisms to maintain genomic stability under various environmental conditions . NE1667, as a putative Holliday junction resolvase, would play a critical role in homologous recombination-based DNA repair.

The genome of Nitrosomonas europaea contains complex repetitive elements constituting approximately 5% of the genome, including 85 predicted insertion sequence elements in eight different families . These repetitive elements may increase the likelihood of recombination events requiring resolution by NE1667. Additionally, the organism's strategy for iron accumulation involves several classes of Fe receptors with more than 20 genes devoted to these receptors . Iron metabolism can generate reactive oxygen species that damage DNA, potentially increasing the need for efficient DNA repair mechanisms.

Research examining the expression patterns of NE1667 under different growth conditions or stress factors would provide insights into its role in maintaining genomic integrity in this specialized bacterium.

What are the recommended protocols for expression and purification of recombinant NE1667?

Based on standard approaches for recombinant protein production, the following protocol framework is recommended for NE1667:

• Gene Cloning:

  • Amplify the NE1667 gene from Nitrosomonas europaea genomic DNA

  • Clone into an expression vector with appropriate tags (His-tag recommended for purification)

  • Verify the sequence integrity before expression

• Expression System Selection:

  • E. coli BL21(DE3) or similar strains are typically suitable for recombinant protein expression

  • Consider codon optimization if expression levels are low

  • Test expression in different media (LB, TB, auto-induction) and temperatures (16°C, 25°C, 37°C)

• Purification Strategy:

  • Lyse cells in buffer containing appropriate protease inhibitors

  • Conduct initial purification using immobilized metal affinity chromatography (IMAC)

  • Further purify using ion exchange and/or size exclusion chromatography

  • Assess purity by SDS-PAGE and activity by functional assays

• Protein Storage:

  • Store in buffer containing glycerol (20-30%) at -80°C for long-term storage

  • For improved stability, consider lyophilization which extends shelf life to 12 months at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

What assays can be used to characterize the enzymatic properties of NE1667?

Several complementary assays can be employed to characterize the enzymatic properties of NE1667:

• Synthetic Holliday Junction Cleavage Assay:

  • Prepare synthetic four-way junctions using oligonucleotides with fluorescent or radioactive labels

  • Incubate with purified NE1667 under various conditions

  • Analyze cleavage products by denaturing polyacrylamide gel electrophoresis

  • Quantify to determine reaction kinetics and substrate specificity

• Cruciform Plasmid Cleavage Assay:

  • Use plasmids containing inverted repeats that form cruciforms under supercoiling

  • Analyze products by agarose gel electrophoresis to distinguish between coordinated and uncoordinated cleavage

• DNA Binding Assays:

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

  • Fluorescence anisotropy to measure binding kinetics

  • Surface plasmon resonance for real-time binding analysis

• Structure-Function Analysis:

  • Generate site-directed mutants of conserved residues

  • Assess the effect on activity using the above assays

  • Compare results with known structure-function relationships of other HJ resolvases

These methods together provide a comprehensive characterization of the enzyme's activity, specificity, and mechanism.

How can NE1667 contribute to our understanding of bacterial DNA repair mechanisms?

Studying NE1667 can provide valuable insights into DNA repair mechanisms in bacteria, particularly in species with specialized metabolisms like Nitrosomonas europaea. As a Holliday junction resolvase, NE1667 represents an important component of homologous recombination pathways that are critical for maintaining genomic integrity.

Comparative analysis of NE1667 with other bacterial Holliday junction resolvases could reveal adaptations specific to the ecological niche and metabolic characteristics of Nitrosomonas europaea. The study of NE1667 may help elucidate how DNA repair mechanisms have evolved in bacteria with different lifestyles and environmental adaptations.

What are the potential biotechnological applications of NE1667?

Holliday junction resolvases have potential applications in biotechnology, particularly in genetic engineering and DNA manipulation techniques. Potential applications of NE1667 include:

• Development of new molecular biology tools for DNA manipulation

• Enhancement of homologous recombination efficiency in genetic engineering

• Potential use in targeted genetic modification systems

• Application in structural studies of DNA recombination intermediates

The specificity and activity profiles of NE1667 would need to be thoroughly characterized to assess its suitability for these applications. Comparison with existing tools and technologies would be essential to identify unique advantages that NE1667 might offer.

What are the key research gaps in our understanding of NE1667?

Despite the information available on Holliday junction resolvases in general, several knowledge gaps remain regarding NE1667 specifically:

• Detailed structural characterization of NE1667, including potential unique domains

• Biochemical properties and substrate specificity compared to other HJRs

• Expression patterns and regulation in Nitrosomonas europaea

• Potential interactions with other components of the DNA repair machinery

• Evolutionary relationships with HJRs from other bacterial species

• Specific adaptations related to the unique lifestyle of Nitrosomonas europaea

Addressing these gaps would require a combination of structural biology, biochemistry, molecular biology, and comparative genomics approaches.