Recombinant Staphylococcus aureus DNA polymerase III subunit alpha (dnaE), partial

What is the role of DNA polymerase III subunit alpha (dnaE) in Staphylococcus aureus?

DNA polymerase III subunit alpha (dnaE) in Staphylococcus aureus serves as the core catalytic component of DNA polymerase III holoenzyme, which is the primary enzyme complex responsible for chromosomal DNA replication. This subunit possesses the 5′→3′ polymerase activity (EC 2.7.7.7) that synthesizes new DNA strands using the parental DNA as a template . In S. aureus, dnaE works in conjunction with other subunits, including the β-clamp, which enhances processivity during DNA synthesis and interacts with various proteins involved in DNA metabolism . The dnaE subunit plays a crucial role in maintaining genomic integrity and is essential for bacterial survival and pathogenicity.

How is recombinant S. aureus dnaE typically expressed and purified?

Recombinant S. aureus dnaE is typically expressed in E. coli expression systems, which provide high yields of soluble protein . The methodology involves:

• Cloning: The dnaE gene from S. aureus (often strain Mu50/ATCC 700699) is amplified by PCR and cloned into an appropriate expression vector with an affinity tag.

• Expression: The recombinant plasmid is transformed into E. coli expression strains, and protein expression is induced under optimized conditions.

• Purification: The expressed protein is purified using affinity chromatography, typically based on the tag incorporated into the recombinant construct. The final purity is generally >85% as confirmed by SDS-PAGE .

• Storage: The purified protein can be stored in solution at -20°C/-80°C (shelf life approximately 6 months) or in lyophilized form at -20°C/-80°C (shelf life approximately 12 months) .

• Reconstitution: For lyophilized protein, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, with 5-50% glycerol added for long-term storage .

What DNA repair pathways involve DNA polymerase III in S. aureus?

DNA polymerase III in S. aureus participates in several DNA repair pathways:

• Base Excision Repair (BER): DNA polymerase III works in conjunction with other enzymes like AP endonucleases (such as Nfo) to repair damaged DNA bases. S. aureus contains homologues of Nfo, RecJ, PolX, and Pol I that participate in this pathway .

• SOS Response: During significant DNA damage, the SOS response is activated, which involves RecA binding to single-stranded DNA, leading to LexA self-cleavage and derepression of SOS genes. DNA polymerase III plays a role in replicating DNA during this stress response .

• Oxidative Damage Repair: S. aureus must repair oxidative DNA damage, particularly 8-oxoguanine (8-oxoG) lesions that can cause G-to-T transversions. DNA polymerase III, along with specialized repair enzymes, helps maintain genomic integrity under oxidative stress .

Unlike E. coli and B. subtilis, S. aureus has a more limited SOS regulon with fewer genes involved, but still maintains the core components including recA, lexA, and genes encoding DNA repair proteins .

How can I verify the activity of recombinant S. aureus dnaE in vitro?

To verify the activity of recombinant S. aureus dnaE in vitro, researchers typically employ the following methodological approaches:

• DNA Polymerase Activity Assay: Measure the incorporation of radiolabeled or fluorescently labeled nucleotides into a DNA template. This can be quantified by measuring the amount of labeled product formed over time.

• Processivity Assays: Evaluate the ability of dnaE (in conjunction with the β-clamp) to synthesize long stretches of DNA without dissociating from the template by analyzing product length distributions.

• Primer Extension Assays: Assess the ability of dnaE to extend primers annealed to single-stranded DNA templates using gel electrophoresis to visualize the extension products.

• Fidelity Assays: Determine the accuracy of nucleotide incorporation by measuring error rates during DNA synthesis, which can be assessed through sequencing of synthesized products.

• Interaction Studies: Examine the interaction of dnaE with other components of the replisome, particularly the β-clamp, using techniques such as pull-down assays, surface plasmon resonance, or yeast two-hybrid systems .

How do mutations in S. aureus dnaE affect antibiotic resistance development?

Mutations in S. aureus dnaE can significantly impact antibiotic resistance development through several mechanisms:

• Mutator Phenotypes: Alterations in dnaE that reduce the fidelity of DNA replication can lead to increased mutation rates throughout the genome, accelerating the evolution of antibiotic resistance. This is particularly relevant given the role of DNA polymerase III in maintaining replication fidelity.

• SOS Response Modulation: Since dnaE functions within the context of the SOS response, mutations affecting its regulation or activity can alter how S. aureus responds to DNA-damaging antibiotics. The SOS response in S. aureus differs from E. coli and B. subtilis, with a more limited set of genes (approximately 16 compared to 43 and 63, respectively) .

• Stress-Induced Mutagenesis: Under antibiotic stress, alterations in dnaE function can influence error-prone DNA synthesis. In S. aureus, error-prone DNA repair is performed by UmuC during the SOS response, which works in conjunction with DNA polymerase III .

• Recombination Rates: Changes in dnaE activity can affect homologous recombination frequencies, potentially influencing the acquisition of resistance genes through horizontal gene transfer.

Methodologically, researchers investigating these phenomena typically employ:

• Fluctuation analysis to measure mutation rates

• Whole-genome sequencing to identify secondary mutations arising from dnaE variants

• In vitro biochemical assays comparing wild-type and mutant dnaE activities

• Animal infection models to assess the in vivo consequences of dnaE mutations

What are the structural and functional differences between S. aureus dnaE and its homologs in other bacterial species?

S. aureus dnaE exhibits both conserved features and distinct differences compared to homologs in other bacterial species:

Studying these differences requires:

• Comparative sequence and structural analysis using bioinformatics

• Heterologous expression systems to examine the functionality of dnaE from different species

• Chimeric protein construction to identify domain-specific functions

• Crystallography or cryo-EM studies to resolve structural differences

• Biochemical assays comparing enzymatic properties across species

How does the β-clamp interaction with dnaE influence DNA repair and replication in S. aureus?

The interaction between the β-clamp and dnaE is critical for DNA replication and repair in S. aureus, with several important functional implications:

• Processivity Enhancement: The β-clamp forms a ring around DNA and tethers dnaE to the template, dramatically increasing processivity from tens of nucleotides to thousands of nucleotides per binding event.

• Protein Recruitment Hub: Beyond its interaction with dnaE, the β-clamp serves as a platform for recruiting various DNA repair proteins. Recent research has shown that the C-terminal face of the β-clamp interacts with multiple partners, including AP endonuclease Nfo .

• Conserved Interaction Motif: The interaction between dnaE and the β-clamp occurs through a conserved region in the C-terminal portion of the β-clamp. Specifically, the sequence 373PIR375 in S. aureus β-clamp is crucial, with the proline residue playing a particularly important role .

• Effect of Mutations: Mutations in the β-clamp, such as P373A/I374D/R375A, disrupt its ability to stimulate AP endonuclease Nfo activity, highlighting the importance of this interaction in coordinating DNA repair processes .

• Regulatory Role: The β-clamp serves as a central coordinator that links DNA replication and repair, ensuring that these processes are properly synchronized during normal growth and under stress conditions.

Methodologically, this interaction can be studied through:

• Site-directed mutagenesis of the interaction interface

• Protein-protein interaction assays (pull-downs, SPR, etc.)

• Enzymatic assays with wild-type and mutant proteins

• In vivo studies examining the phenotypic effects of disrupting this interaction

What experimental design considerations are crucial for studying S. aureus dnaE in host-pathogen interaction models?

When designing experiments to study S. aureus dnaE in host-pathogen interaction models, several critical factors must be considered:

• Block Randomization: Implement proper experimental design with block randomization of case/control status, site, and DNA extraction method to avoid confounding factors that could lead to spurious associations . This is particularly important as approximately 95% of studies have major problems with experimental design, primarily related to randomization issues .

• Host DNA Damage Assessment: S. aureus is known to induce DNA damage in host cells through the production of reactive oxygen species (ROS) that promote oxidation of guanine to form 8-oxoG . When studying dnaE's role in pathogenesis, researchers should include methods to assess:

  • Histone H2AX phosphorylation (a marker of DNA double-strand breaks)

  • ROS levels and oxidative DNA damage markers

  • Host DNA repair pathway activation (e.g., ATM kinase signaling)

• Extracellular DNA Considerations: S. aureus secretes immunomodulatory RNA and DNA molecules, some protected by membrane vesicles (MVs) . This extracellular DNA can confound host-pathogen interaction studies and should be controlled for by:

  • Purifying MVs to reduce exogenous DNA from broken cells

  • Distinguishing between bacterial DNA integrated into host cells versus extracellular DNA

  • Using DNase treatments to eliminate extracellular DNA contamination

• Species-Specific Assays: Develop species-specific and ubiquitous DNA-based assays to accurately identify and quantify S. aureus in experimental samples . These assays should:

  • Be specific for S. aureus (test against other staphylococcal species)

  • Be sensitive enough to detect low bacterial loads

  • Be suitable for different sample types (tissue cultures, blood cultures, etc.)

  • Be capable of distinguishing bacterial from host DNA

• Consideration of Virulence Factors: Account for the effects of virulence factors like phenol-soluble modulins (PSMα 1–4) that can damage host DNA, and lipoproteins that may counteract this damage .

How can recombinant DNA technology be optimized for studying S. aureus dnaE structure-function relationships?

Optimizing recombinant DNA technology for studying S. aureus dnaE structure-function relationships requires sophisticated approaches:

• Expression System Selection:

  • Prokaryotic systems (typically E. coli) offer high yields but may present folding challenges for large proteins like dnaE

  • Cell-free expression systems can be advantageous for potentially toxic proteins

  • Expression conditions should be systematically optimized for temperature, induction time, and media composition

• Protein Engineering Strategies:

  • Design truncated constructs to express functional domains separately

  • Introduce affinity tags that minimally impact function (His-tag, Strep-tag)

  • Create fusion proteins with solubility enhancers (MBP, SUMO) that can be removed post-purification

  • Implement site-directed mutagenesis to examine specific residues in catalytic sites or protein interaction interfaces

• Structural Analysis Methods:

  • X-ray crystallography for high-resolution static structures

  • Cryo-electron microscopy for visualizing larger complexes (e.g., dnaE with β-clamp)

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamics and interactions

  • Small-angle X-ray scattering (SAXS) for solution-state conformational information

• Functional Assessment:

  • Develop high-throughput activity assays for mutational scanning

  • Employ pre-steady-state kinetics to dissect individual steps in the catalytic cycle

  • Use fluorescently labeled substrates for real-time monitoring of activity

  • Implement single-molecule approaches to examine dynamics without ensemble averaging

• In vitro Reconstitution:

  • Assemble minimal replisomes with purified components to study dnaE in context

  • Utilize DNA substrates with specific structures (e.g., fork templates, damaged DNA)

  • Combine with other DNA repair proteins to examine coordinated activities

These approaches collectively enable detailed structure-function analysis that can reveal mechanisms of dnaE activity, regulation, and interactions with other replisome components .

What are the optimal storage and handling conditions for recombinant S. aureus dnaE?

The optimal storage and handling conditions for recombinant S. aureus DNA polymerase III subunit alpha (dnaE) are critical for maintaining its activity and stability:

• Storage Temperature:

  • For liquid formulations: -20°C to -80°C for up to 6 months

  • For lyophilized formulations: -20°C to -80°C for up to 12 months

• Working Aliquots:

  • Store at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles which can degrade protein activity

• Reconstitution Protocol:

  • Briefly centrifuge vials prior to opening to bring contents to the bottom

  • Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% is typical) for long-term storage

• Buffer Considerations:

  • pH stability is typically optimal between 7.0-8.0

  • Include reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

  • Consider adding metal ion chelators (EDTA) to prevent metal-catalyzed oxidation

  • Protease inhibitors may be necessary in certain applications

• Quality Control Monitoring:

  • Periodically check activity using standardized assays

  • Verify protein integrity by SDS-PAGE

  • Monitor for precipitation or aggregation

  • Document storage time and conditions for each lot

Adherence to these handling procedures ensures maximum retention of enzymatic activity and experimental reproducibility when working with this recombinant protein.

How do different purification strategies affect the activity and stability of recombinant S. aureus dnaE?

Different purification strategies can significantly impact the activity and stability of recombinant S. aureus dnaE:

• Tag Selection Impact:

  • His-tags generally have minimal impact on polymerase activity but may introduce metal ion sensitivity

  • Larger tags (MBP, GST) can enhance solubility but potentially interfere with function

  • Tag position (N- vs C-terminal) matters, as C-terminal tags may disrupt β-clamp interactions

  • Tag removal may be necessary for structural studies or when examining protein-protein interactions

• Purification Method Comparison:

• Buffer Optimization:

  • Addition of stabilizing agents (glycerol, trehalose) improves stability

  • Inclusion of divalent cations (Mg²⁺) maintains structural integrity

  • Optimized salt concentration prevents non-specific interactions

  • Reducing agents protect catalytic cysteine residues

• Source Organism Considerations:

  • Expression in E. coli is standard but can introduce host-specific modifications

  • Expression in B. subtilis might provide more native-like folding for gram-positive proteins

  • Cell-free systems avoid cellular toxicity issues but may yield lower amounts

• Activity Retention Strategies:

  • Co-purification with stabilizing partners (e.g., β-clamp fragments)

  • Rapid purification at lower temperatures (4°C)

  • Addition of nucleic acid substrates to stabilize active conformations

  • Limited proteolysis approaches to identify stable domains

Each purification strategy presents trade-offs between yield, purity, activity, and structural integrity that must be considered based on the intended experimental application.

What analytical methods can detect structural changes in dnaE resulting from site-directed mutagenesis?

Several sophisticated analytical methods can detect and characterize structural changes in dnaE resulting from site-directed mutagenesis:

• Spectroscopic Techniques:

  • Circular Dichroism (CD): Monitors secondary structure changes (α-helices, β-sheets)

  • Fluorescence Spectroscopy: Detects tertiary structure alterations via intrinsic tryptophan fluorescence

  • Fourier Transform Infrared Spectroscopy (FTIR): Provides information about protein secondary structure

  • Nuclear Magnetic Resonance (NMR): Offers atomic-level structural information for smaller domains

• Thermal and Chemical Stability Assessments:

  • Differential Scanning Calorimetry (DSC): Measures thermodynamic parameters of unfolding

  • Thermal Shift Assays: Monitors protein unfolding via hydrophobic dye binding

  • Chemical Denaturation: Quantifies stability changes through unfolding curves

  • Limited Proteolysis: Identifies regions with altered structural protection

• Hydrodynamic Methods:

  • Size Exclusion Chromatography (SEC): Detects changes in oligomeric state or shape

  • Analytical Ultracentrifugation (AUC): Provides information on molecular weight and shape

  • Dynamic Light Scattering (DLS): Measures hydrodynamic radius and size distribution

  • Small-Angle X-ray Scattering (SAXS): Generates low-resolution structural models in solution

• High-Resolution Structural Techniques:

  • X-ray Crystallography: Provides atomic-level structures, ideal for comparing wild-type and mutant proteins

  • Cryo-Electron Microscopy: Visualizes larger complexes and conformational states

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Maps solvent accessibility changes

  • Cross-linking Mass Spectrometry (XL-MS): Identifies altered spatial relationships between domains

• Computational Approaches:

  • Molecular Dynamics Simulations: Predicts dynamic behavior changes due to mutations

  • Homology Modeling: Generates structural models based on similar proteins

  • Normal Mode Analysis: Identifies altered flexibility patterns

  • In silico Alanine Scanning: Predicts effects of mutations on stability and interactions

When studying mutations in conserved regions like the β-clamp interaction motif (373PIR375) , these methods can reveal how structural changes correlate with functional alterations, providing deeper insights into structure-function relationships.

How does the DNA repair function of S. aureus dnaE compare with other bacterial pathogens?

The DNA repair function of S. aureus dnaE exhibits both similarities and distinct differences when compared to other bacterial pathogens:

• SOS Response System Comparison:

• DNA Repair Pathway Variations:

  • Base Excision Repair (BER): S. aureus contains homologues of Nfo, RecJ, PolX, and Pol I , similar to other pathogens, but lacks certain enzymes present in other species

  • Methyl-Directed Mismatch Repair: Less well-characterized in S. aureus compared to E. coli

  • Oxidative Damage Repair: Critical for S. aureus survival in the oxidative environment of host immune cells

  • Direct Repair: S. aureus lacks homologues of ada and alkB genes that are present in E. coli and B. subtilis

• Interaction with Host Defenses:

  • S. aureus dnaE functions within a repair system that must counter host-generated ROS, which can cause DNA damage including 8-oxoG formation

  • Unlike some pathogens that primarily reside inside cells, S. aureus alternates between extracellular and intracellular environments, requiring adaptable DNA repair mechanisms

  • S. aureus produces PSMα peptides that damage host DNA, creating a competitive advantage

• β-clamp Interactions:

  • The interaction between dnaE and the β-clamp via conserved motifs (373PIR375 in S. aureus) is fundamental across bacteria but shows species-specific variations

  • Research has shown that in S. aureus, the β-clamp interacts with AP endonuclease Nfo, suggesting a more direct role in DNA repair than previously thought

• Methodological Approaches for Comparative Studies:

  • Heterologous complementation assays to assess functional conservation

  • Chimeric protein construction to identify domain-specific functions

  • Bioinformatic analysis of repair gene clusters and regulatory networks

  • Comparative genomics to identify species-specific adaptations in DNA repair systems

What are the implications of S. aureus dnaE research for developing novel antimicrobial strategies?

Research on S. aureus dnaE offers several promising avenues for developing novel antimicrobial strategies:

• Direct Targeting of DNA Replication:

  • dnaE represents an essential bacterial enzyme with no human homolog, making it an attractive antibiotic target

  • Structure-based drug design focused on the catalytic site could yield specific inhibitors

  • Targeting the dnaE-β-clamp interaction represents another approach, as disrupting this interaction compromises DNA replication fidelity and efficiency

  • The conserved nature of DNA polymerase III across bacterial species offers the potential for broad-spectrum activity

• Exploiting DNA Repair Deficiencies:

  • Compounds that generate DNA damage specifically repaired by dnaE-dependent pathways could be selectively toxic to S. aureus

  • Combinatorial approaches targeting both dnaE and other DNA repair pathways could enhance killing efficiency

  • The interplay between dnaE and the SOS response suggests that SOS inhibitors could potentiate conventional antibiotics

• Sensitization to Host Immune Defenses:

  • Inhibition of dnaE could sensitize S. aureus to oxidative damage from neutrophils and macrophages

  • Since S. aureus must repair DNA damage caused by ROS during infection , compromising this ability through dnaE targeting would reduce virulence

  • The balance between bacterial DNA damage and host DNA damage (caused by factors like PSMα) could be exploited therapeutically

• Antibiotic Resistance Considerations:

  • Targeting dnaE might slow the development of resistance to other antibiotics by reducing mutation rates

  • The essential nature of dnaE might create a higher barrier to resistance development

  • Understanding how mutations in dnaE contribute to hypermutator phenotypes could help predict and counter resistance evolution

• Methodological Approaches for Drug Development:

  • High-throughput screening of compound libraries against purified dnaE

  • Fragment-based drug discovery to identify initial chemical matter

  • Structure-based virtual screening using crystal structures or homology models

  • Whole-cell assays to identify compounds that can penetrate the cell wall and remain active

  • In vivo infection models to validate target engagement and efficacy

The pursuit of these strategies requires detailed understanding of dnaE structure-function relationships and its interactions within the replisome complex, highlighting the importance of basic research in this area .

What emerging technologies might enhance our understanding of S. aureus dnaE in the context of antibiotic resistance?

Several cutting-edge technologies are poised to revolutionize our understanding of S. aureus dnaE in antibiotic resistance:

• CRISPR-Cas9 Genome Editing:

  • Precise engineering of dnaE variants in S. aureus to study the effects of specific mutations on replication fidelity and antibiotic resistance

  • Creation of conditional knockdowns to identify genetic interactions with other DNA repair pathways

  • Development of base editors to introduce specific mutations without double-strand breaks

  • Implementation of CRISPR interference (CRISPRi) for tunable repression of dnaE expression

• Single-Cell Technologies:

  • Single-cell RNA sequencing to capture heterogeneity in dnaE expression during antibiotic exposure

  • Time-lapse microscopy with fluorescent reporters to visualize dnaE activity in real-time

  • Single-cell proteomics to assess protein-level changes in the DNA replication machinery

  • Microfluidic devices to track mutation rates at the single-cell level under antibiotic pressure

• Structural Biology Advances:

  • Cryo-electron tomography to visualize intact replisomes within bacterial cells

  • Integrative structural biology combining multiple techniques (X-ray, cryo-EM, NMR, HDX-MS) for complete structural models

  • Time-resolved structural methods to capture conformational changes during catalysis

  • AlphaFold2 and related AI approaches to predict structures of dnaE in complex with other replisome components

• Systems Biology Approaches:

  • Multi-omics integration combining transcriptomics, proteomics, and metabolomics to map dnaE's role in antibiotic response networks

  • Network analysis to identify synthetic lethal interactions with dnaE that could be exploited therapeutically

  • Mathematical modeling of mutation rates and resistance development incorporating dnaE function

  • Genome-scale metabolic models to predict how dnaE inhibition affects bacterial physiology

• Advanced Imaging Technologies:

  • Super-resolution microscopy (STORM, PALM) to visualize replisome dynamics

  • Correlative light and electron microscopy (CLEM) to link dnaE localization with cellular ultrastructure

  • Expansion microscopy to physically enlarge bacterial cells for improved imaging resolution

  • DNA damage sensors to visualize repair processes in real-time

These emerging technologies will help address fundamental questions about how dnaE mutations influence antibiotic resistance development, potentially leading to new therapeutic strategies targeting DNA replication and repair in S. aureus.

How might experimental design be improved to better study the role of dnaE in S. aureus pathogenesis?

Experimental design for studying dnaE in S. aureus pathogenesis can be significantly improved through several methodological refinements:

• Rigorous Randomization and Controls:

  • Implement block randomization involving case/control status, site, and DNA extraction method to eliminate batch effects

  • Include multiple negative and positive controls for each experimental condition

  • Blind researchers to sample identity during processing and analysis

  • Perform power calculations to ensure adequate sample sizes for detecting biologically meaningful effects

• In vivo Infection Models with Enhanced Relevance:

  • Develop conditional expression systems for dnaE variants that can be activated during infection

  • Utilize humanized mouse models that better recapitulate the human immune environment

  • Implement tissue-specific infection models that mimic common S. aureus infection sites (skin, bone, heart)

  • Track bacterial population dynamics in vivo using barcoding approaches to monitor selection

• Advanced Host-Pathogen Interaction Studies:

  • Account for S. aureus extracellular DNA/RNA that may confound host-pathogen studies

  • Develop assays to distinguish between bacterial DNA integrated into host cells versus extracellular DNA

  • Implement dual RNA-seq to simultaneously profile host and pathogen transcriptomes during infection

  • Analyze host DNA damage responses (H2AX phosphorylation, 8-oxoG formation) in relation to bacterial dnaE activity

• Improved Species-Specific Detection Methods:

  • Develop highly specific and sensitive PCR-based assays targeting the 442-bp DNA fragment identified as S. aureus-specific

  • Implement droplet digital PCR for absolute quantification of bacterial loads

  • Develop multiplex assays that simultaneously detect S. aureus and assess antimicrobial resistance markers

  • Utilize CRISPR-based detection systems for rapid identification in complex samples

• Integrative Multi-Level Analysis:

  • Combine genomic, transcriptomic, and proteomic analyses to build comprehensive models of dnaE function

  • Track mutations arising in dnaE and other genes during infection using deep sequencing

  • Correlate bacterial genotype with host immune responses and clinical outcomes

  • Implement systems biology approaches to model the complex interplay between bacterial DNA repair and host immunity

• Standardized Reporting and Data Sharing:

  • Adopt MIAME/MINSEQE-like standards for experimental reporting

  • Establish repositories for sharing raw data and analytical pipelines

  • Implement electronic lab notebooks for enhanced reproducibility

  • Develop community standards for S. aureus strain collections and nomenclature

These improvements would address current limitations in experimental design that have led to problems in approximately 95% of studies , enabling more robust and reproducible research on dnaE's role in S. aureus pathogenesis.