Recombinant Legionella pneumophila subsp. pneumophila Putative 3-methyladenine DNA glycosylase (lpg0866)

What is Recombinant Legionella pneumophila subsp. pneumophila Putative 3-methyladenine DNA glycosylase?

Recombinant Legionella pneumophila subsp. pneumophila Putative 3-methyladenine DNA glycosylase (lpg0866) is a recombinant protein derived from Legionella pneumophila subspecies pneumophila strain Philadelphia 1 (ATCC 33152/DSM 7513). It functions as a DNA glycosylase (EC 3.2.2.-) and is implicated in the base excision repair (BER) pathway. The protein is expressed recombinantly in E. coli and comprises 183 amino acids in its full-length form. It is identified by UniProt accession number Q5ZX66 and is typically supplied with product code CSB-EP720075LDL for research applications . The protein likely plays a role in removing alkylated bases, particularly 3-methyladenine, from damaged DNA, initiating the base excision repair process.

What is the role of DNA glycosylases in base excision repair?

DNA glycosylases, such as lpg0866, are enzymes that initiate the base excision repair (BER) pathway, which is responsible for correcting various types of DNA damage including oxidation, alkylation, or abasic sites. These enzymes recognize and remove damaged bases by cleaving the N-glycosidic bond between the base and the deoxyribose sugar, leaving an apurinic/apyrimidinic (AP) site. This non-instructive AP site is then processed through either of two BER sub-pathways: short-patch BER (SP-BER), which replaces just one nucleotide, or long-patch BER (LP-BER), which replaces more than one nucleotide . The selection of the appropriate sub-pathway depends on various factors including the type of damage, the cell cycle phase, and the availability of specific repair factors. DNA glycosylases like lpg0866 often demonstrate specificity for particular types of base damage, contributing to the precision of the DNA repair mechanism.

How should lpg0866 be stored and handled for optimal stability?

For optimal stability and activity retention, Recombinant Legionella pneumophila subsp. pneumophila Putative 3-methyladenine DNA glycosylase (lpg0866) should be stored according to specific conditions depending on its formulation. The liquid form generally has a shelf life of approximately 6 months when stored at -20°C/-80°C, while the lyophilized form can maintain stability for up to 12 months at the same temperature range . To prevent protein degradation due to freeze-thaw cycles, it is recommended not to repeatedly freeze and thaw the protein. Working aliquots can be stored at 4°C for up to one week to minimize degradation. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) prior to aliquoting for long-term storage at -20°C/-80°C . This approach preserves the protein's structural integrity and enzymatic activity for experimental use.

How does lpg0866 participate in different BER sub-pathways during DNA repair?

Recombinant Legionella pneumophila subsp. pneumophila Putative 3-methyladenine DNA glycosylase (lpg0866) likely participates in the base excision repair (BER) pathway, which can proceed through either short-patch BER (SP-BER) or long-patch BER (LP-BER) sub-pathways. Research indicates that the choice between these sub-pathways is influenced by the type of DNA damage. For instance, studies with 8-oxo-7,8-dihydroguanine (8-oxoG) lesions have shown that LP-BER occurs in 55-80% of cases with repair patches of 2-6 nucleotides, while synthetic abasic sites are processed by LP-BER in 80-100% of cases with patches of 6-12 nucleotides . As a putative 3-methyladenine DNA glycosylase, lpg0866 would initiate repair by removing alkylated bases, creating an AP site that would then be further processed. The enzyme's activity might influence whether the damage is subsequently handled by SP-BER or LP-BER, depending on factors such as the structural changes induced in the DNA backbone and the recruitment of downstream repair factors. Understanding this mechanism requires analysis of protein-protein interactions and repair pathway kinetics in the context of specific DNA lesions.

What structural features of lpg0866 contribute to its substrate specificity?

While the complete three-dimensional structure of lpg0866 has not been fully characterized in the provided search results, its function as a putative 3-methyladenine DNA glycosylase suggests specific structural elements that would contribute to its substrate recognition and catalytic activity. Based on homology to other DNA glycosylases, lpg0866 likely contains a nucleotide-flipping mechanism that allows it to recognize and excise damaged bases from the DNA double helix. The protein sequence (MRKLLRPFYERDTVLVAKELLGKYLVHHDGLEEKIGRIVEVEAYLGQHDLACHSSKGLTKRTKVMFGPAGYAYVYLIYGMYYCMNVVTEKEGIGSALIVRALEP IKNIQDRTHGPGLLSKAMRIDSKLNHRDLLSNDFYIAEPNSPTDFTIIEKPRIGVHYAKEWANELLRFYIKDNPYISKT) contains regions that may form a catalytic pocket specific for alkylated bases, particularly 3-methyladenine . Structural analysis using X-ray crystallography or cryo-electron microscopy would be necessary to definitively identify these regions. Additionally, comparative structural analysis with related glycosylases could provide insights into conserved domains that determine substrate specificity. Site-directed mutagenesis experiments targeting specific amino acid residues would help elucidate which regions are essential for substrate recognition versus catalytic activity.

How does lpg0866 compare with other bacterial DNA glycosylases in terms of activity and specificity?

A comprehensive comparative analysis of lpg0866 with other bacterial DNA glycosylases would require examination of enzymatic parameters and substrate preferences. While specific kinetic data for lpg0866 is not provided in the search results, its classification as a putative 3-methyladenine DNA glycosylase suggests similarity to other glycosylases that recognize and remove alkylated bases. DNA glycosylases across bacterial species often share conserved structural motifs despite relatively low sequence identity, reflecting evolutionary pressure to maintain specific catalytic functions. Comparisons should include analysis of:

Comprehensive biochemical characterization including substrate range testing, enzyme kinetics, and inhibition studies would be necessary to fully elucidate how lpg0866 compares with other bacterial DNA glycosylases.

What are the optimal conditions for assessing lpg0866 enzymatic activity in vitro?

To optimize in vitro enzymatic activity assays for Recombinant Legionella pneumophila subsp. pneumophila Putative 3-methyladenine DNA glycosylase (lpg0866), researchers should consider several key parameters. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol as recommended for stability . A typical DNA glycosylase activity assay would involve:

• Substrate preparation: Synthetic oligonucleotides containing specific lesions (e.g., 3-methyladenine, or other alkylated bases) at defined positions.

• Reaction buffer composition: Usually containing Tris-HCl (pH 7.5-8.0), NaCl (50-150 mM), EDTA (1-5 mM), DTT (1-2 mM), and BSA (0.1-0.5 mg/mL).

• Temperature and time: Incubation at 37°C for 30-60 minutes, with time course measurements to determine optimal reaction times.

• Product analysis: Denaturing PAGE to visualize cleaved products, or HPLC analysis of released bases.

Since lpg0866 has a purity of >85% by SDS-PAGE , researchers should consider this when calculating enzyme concentrations for kinetic studies. Control experiments should include heat-inactivated enzyme and substrates without lesions. For quantitative analysis, a standard curve using known concentrations of product oligonucleotides or free bases would enable precise activity measurements. These methodological details should be optimized for lpg0866 specifically, as conditions may differ from those used for other DNA glycosylases.

How can researchers effectively express and purify functional lpg0866?

For efficient expression and purification of functional lpg0866, researchers should consider the following protocol based on standard recombinant protein techniques and the specific information about this protein:

• Expression system: According to the search results, lpg0866 is expressed in E. coli , which suggests that standard E. coli expression systems (e.g., BL21(DE3), Rosetta) are suitable. The gene should be codon-optimized for E. coli expression.

• Vector selection: A vector containing an appropriate promoter (e.g., T7) and a fusion tag for purification. The search results mention that "Tag type will be determined during the manufacturing process" , indicating flexibility in tag selection.

• Culture conditions:

  • Medium: LB or 2XYT supplemented with appropriate antibiotics

  • Induction: 0.1-1.0 mM IPTG when OD600 reaches 0.6-0.8

  • Temperature: 16-25°C for 16-20 hours (lower temperatures may enhance soluble protein yield)

• Purification strategy:

  • Lysis buffer: Typically containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole (for His-tagged proteins), protease inhibitors

  • Affinity chromatography: Based on the selected tag

  • Size exclusion chromatography: To ensure homogeneity and remove aggregates

  • Final buffer: Compatible with storage conditions (with 5-50% glycerol)

• Quality control:

  • SDS-PAGE to confirm purity (target >85% as indicated in the search results )

  • Activity assay to confirm functionality

  • Mass spectrometry to verify protein identity

This methodological approach should yield functional lpg0866 suitable for experimental applications, considering the protein's full-length nature (183 amino acids) and specific storage requirements.

What experimental approaches can be used to study lpg0866 interaction with damaged DNA?

Several experimental approaches can be employed to study the interaction between lpg0866 and damaged DNA substrates:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified lpg0866 with fluorescently or radioactively labeled DNA oligonucleotides containing specific lesions

  • Analyze protein-DNA complexes by native PAGE

  • Determine binding affinities (Kd) through titration experiments

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Immobilize either the protein or DNA substrate on a sensor chip

  • Measure real-time binding and dissociation kinetics

  • Calculate kon, koff, and Kd values for different damaged DNA substrates

• Fluorescence Anisotropy:

  • Use fluorescently labeled damaged DNA substrates

  • Monitor changes in anisotropy upon protein binding

  • Determine binding constants and specificity

• X-ray Crystallography or Cryo-EM:

  • Crystallize or prepare lpg0866 in complex with damaged DNA

  • Determine three-dimensional structure

  • Identify specific amino acid-nucleotide interactions

• Molecular Dynamics Simulations:

  • Use the protein sequence provided in the search results to model lpg0866 structure

  • Simulate interactions with damaged DNA substrates

  • Identify key interaction points and conformational changes

• Base Excision Activity Assays:

  • Design DNA substrates with different lesions at specific positions

  • Measure cleavage rates using denaturing PAGE

  • Compare kinetic parameters (kcat, Km) for different substrates

These methodological approaches would provide comprehensive insights into how lpg0866 recognizes, binds, and processes damaged DNA, which is essential for understanding its role in the BER pathway.

How should researchers interpret enzymatic activity data for lpg0866 in different experimental contexts?

When interpreting enzymatic activity data for lpg0866, researchers should consider multiple factors that influence the protein's function across different experimental contexts. First, baseline activity measurements should be established using standardized substrates containing 3-methyladenine or other alkylated bases. Typical enzymatic parameters to measure include:

When comparing lpg0866 activity across experimental contexts, researchers should normalize data to account for variations in protein concentration, purity (which should be >85% by SDS-PAGE ), and assay conditions. For in vivo versus in vitro comparisons, researchers should consider that the base excision repair pathway involves multiple proteins working in concert. Studies have shown that for certain DNA lesions, long-patch BER (LP-BER) can occur in 55-100% of cases, depending on the specific damage . Therefore, the activity of lpg0866 may differ significantly in cellular environments compared to purified systems.

What are the key considerations when analyzing lpg0866 involvement in long-patch versus short-patch BER?

When analyzing the involvement of lpg0866 in long-patch versus short-patch BER pathways, researchers should consider several key factors based on the current understanding of BER mechanisms. Research has demonstrated that the choice between these sub-pathways is significantly influenced by the nature of the DNA lesion. For instance, 8-oxo-7,8-dihydroguanine (8-oxoG) lesions are processed by LP-BER in 55-80% of cases with repair patches spanning 2-6 nucleotides, while synthetic abasic sites undergo LP-BER in 80-100% of cases with longer patches of 6-12 nucleotides .

For rigorous analysis of lpg0866's role in these pathways, researchers should consider:

• Lesion-specific effects: Different alkylated bases may channel repair through different sub-pathways.

• Experimental system design: In vitro versus cellular models may yield different results. The EGFP-based reporter system described in the search results offers a methodological approach to measure repair patch length in vivo.

• Protein interactions: The interaction of lpg0866 with downstream BER factors like AP endonuclease, DNA polymerase β, FEN1, PCNA, and DNA ligase I/III should be evaluated to understand pathway choice.

• Cell cycle dependence: The prevalence of LP-BER versus SP-BER may vary with cell cycle phase.

• Quantitative analysis: Techniques like the stop codon reversion assay described in the search results can provide quantitative data on repair patch length distribution.

When designing experiments, researchers should include appropriate controls and varying distances between the lesion site and marker positions to accurately determine repair patch length, similar to the methodology described for studying 8-oxoG repair .

How can structural data be integrated with functional analyses to better understand lpg0866 mechanism?

Integrating structural and functional data provides a comprehensive understanding of lpg0866's mechanism of action. While specific structural information for lpg0866 is not provided in the search results, researchers can employ the following integrative approach:

• Structure prediction and modeling:

  • Use the complete amino acid sequence provided in the search results for homology modeling

  • Apply molecular dynamics simulations to predict substrate binding sites

  • Identify conserved motifs by comparing with known DNA glycosylase structures

• Mutational analysis guided by structural insights:

  • Design site-directed mutations of predicted catalytic residues

  • Create chimeric proteins with related glycosylases to map functional domains

  • Measure activity changes in mutant proteins to validate structural predictions

• Correlation of structural elements with kinetic parameters:

  • Map substrate specificity determinants by comparing activity on different DNA lesions

  • Identify structural features that influence the rate-limiting step

  • Analyze how structural changes affect the protein's ability to distinguish between damaged and undamaged bases

• Integration with interaction data:

  • Use structural information to predict protein-protein interaction surfaces

  • Verify these predictions with co-immunoprecipitation or yeast two-hybrid studies

  • Determine how these interactions influence BER sub-pathway choice

• Data visualization and integration:

  • Develop 3D models highlighting structure-function relationships

  • Create pathway maps showing how structural elements influence repair process flow

  • Use integrative bioinformatics approaches to combine multiple data types

This integrative approach would enable researchers to build a mechanistic model explaining how the structural features of lpg0866 determine its catalytic activity, substrate specificity, and role in directing repair through either short-patch or long-patch BER pathways.

How can lpg0866 be utilized in developing new DNA repair research methodologies?

Recombinant Legionella pneumophila subsp. pneumophila Putative 3-methyladenine DNA glycosylase (lpg0866) offers several innovative applications for developing new DNA repair research methodologies. As a DNA glycosylase with putative activity against alkylated bases, lpg0866 could be employed in:

• Biosensor development: The protein could be modified with fluorescent tags to create FRET-based sensors that detect conformational changes upon DNA damage recognition, enabling real-time monitoring of repair initiation.

• DNA damage mapping techniques: By combining lpg0866 with next-generation sequencing approaches, researchers could develop methods to map alkylation damage across entire genomes with single-nucleotide resolution.

• BER pathway dissection: The recombinant protein (>85% purity ) could be used in reconstituted repair systems to study pathway choice mechanisms between SP-BER and LP-BER, similar to the approach used for studying 8-oxoG repair .

• Reporter systems: Building on the EGFP-based reporter system described in the search results , lpg0866-specific substrates could be incorporated into cellular reporters to study repair kinetics in living cells.

• Structural biology applications: The availability of the full-length protein (183 amino acids ) enables structural studies to elucidate damage recognition mechanisms, potentially leading to new structure-based assay designs.

• Synthetic biology approaches: lpg0866 could be engineered to recognize novel DNA lesions, creating tools for studying previously inaccessible aspects of DNA damage processing.

These methodological applications would advance our understanding of BER mechanisms and potentially reveal unique aspects of DNA repair in bacterial systems, with possible translation to understanding eukaryotic repair processes as well.

What are the implications of understanding lpg0866 function for studying DNA repair in bacterial pathogens?

Understanding the function of lpg0866 has significant implications for studying DNA repair in Legionella pneumophila and other bacterial pathogens. As a putative 3-methyladenine DNA glycosylase involved in the base excision repair pathway, lpg0866 likely plays a crucial role in helping L. pneumophila maintain genomic integrity, particularly when facing DNA-damaging conditions within host cells or in the environment. Several key implications include:

• Pathogen survival mechanisms: DNA repair systems like BER are essential for bacterial survival during host immune responses that generate DNA-damaging reactive oxygen and nitrogen species. Understanding lpg0866 function could reveal how L. pneumophila persists within macrophages and amoebae.

• Evolution of repair pathways: Comparative analysis of lpg0866 with homologs in other bacterial pathogens could provide insights into the evolution of DNA repair mechanisms and their adaptation to different environmental niches.

• Potential therapeutic targets: DNA repair enzymes represent potential targets for antimicrobial development. Detailed characterization of lpg0866 structure and function could identify unique features that differentiate it from human homologs, enabling selective targeting.

• Stress response coordination: DNA repair is often coordinated with other stress responses in bacterial pathogens. Understanding how lpg0866 activity is regulated in response to environmental stressors could reveal integrated survival strategies.

• Host-pathogen interactions: The activity of DNA repair enzymes like lpg0866 may be modulated during host infection, potentially influencing bacterial virulence and persistence.

Research in this area would benefit from integrating structural, biochemical, and genetic approaches to fully characterize lpg0866 function within the context of L. pneumophila pathogenesis and environmental survival.

What experimental challenges might researchers encounter when studying lpg0866, and how can they be addressed?

Researchers studying lpg0866 may encounter several experimental challenges that require strategic approaches to overcome:

• Protein stability and solubility issues:

  • Challenge: Maintaining enzymatic activity during purification and storage.

  • Solution: Implement the recommended storage conditions with 5-50% glycerol at -20°C/-80°C, and avoid repeated freeze-thaw cycles by preparing working aliquots stored at 4°C for up to one week .

• Substrate preparation complexity:

  • Challenge: Generating DNA substrates with specific alkylated bases at defined positions.

  • Solution: Use synthetic oligonucleotides with site-specific modifications, or enzymatic approaches to introduce damage at specific sites.

• Distinguishing enzymatic activities:

  • Challenge: Differentiating glycosylase activity from associated AP lyase activity.

  • Solution: Design assays that can separately measure base removal and strand cleavage, using appropriate controls with purified AP sites.

• Measuring repair patch length accurately:

  • Challenge: Determining whether SP-BER or LP-BER occurs following base excision.

  • Solution: Adapt the EGFP-based reporter system with stop codons at varying distances from the lesion site, as described for studying 8-oxoG repair .

• Structural characterization difficulties:

  • Challenge: Obtaining high-resolution structural data for lpg0866 alone or in complex with DNA.

  • Solution: Employ a combination of X-ray crystallography, cryo-EM, and computational approaches, potentially using the full protein sequence provided for modeling.

• Cellular context complexity:

  • Challenge: Studying lpg0866 function in the cellular environment where multiple BER factors are present.

  • Solution: Develop cell-based assays in Legionella or heterologous systems, with appropriate genetic manipulations to isolate lpg0866-specific effects.

By anticipating these challenges and implementing the suggested solutions, researchers can develop robust experimental approaches for characterizing lpg0866 function and its role in BER pathways.