Recombinant Staphylococcus aureus DNA polymerase III subunit beta (dnaN)

What is the function of DNA polymerase III subunit beta (dnaN) in S. aureus?

The DNA polymerase III beta subunit (dnaN) in S. aureus functions as the sliding clamp component of the DNA replication machinery. It forms a ring-shaped homodimer that encircles DNA and tethers the polymerase to the template, dramatically increasing processivity during replication. The beta subunit interacts with multiple proteins beyond the core polymerase, serving as a central coordination platform for replication and repair processes. Unlike the alpha subunit (PolIIIα) which performs the actual DNA synthesis, the beta subunit provides structural support and enhances efficiency . This distinction is crucial as the PolIIIα subunit belongs to the C-family of DNA polymerases that are structurally and evolutionarily distinct from eukaryotic replicative polymerases .

What expression systems are most effective for producing recombinant S. aureus dnaN?

• Maintaining cultures at 1×10⁶ cells/mL density prior to transfection

• Co-transfecting expression vectors containing codon-optimized genes using polyethylenimine (PEI) at 7.5 μg/mL ratio

• Incubating at 37°C with 8.0% CO₂ and 120 rpm agitation for approximately 7 days

• Harvesting by centrifugation at 1000 rpm for 5 minutes at 4°C

These parameters may need adjustment for dnaN specifically, but provide a methodological starting point.

How do mutations in S. aureus dnaN affect antibiotic resistance profiles?

Mutations in the S. aureus dnaN gene can significantly impact antibiotic resistance by altering replication fidelity and affecting DNA damage tolerance pathways. The beta clamp interacts with multiple proteins involved in translesion synthesis and mutagenic pathways. While the search results don't directly address dnaN mutations, we can infer from studies on DNA polymerase III that alterations to replication machinery components can contribute to genomic plasticity.

For methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains, differences in replication machinery may contribute to their distinct phenotypes . Experimental approaches to study this relationship should include:

• Site-directed mutagenesis of conserved residues in dnaN

• Phenotypic characterization of mutants for growth rates and antibiotic susceptibility

• Measurement of mutation rates under antibiotic stress

• Assessment of protein-protein interactions with repair polymerases

What purification strategies yield the highest purity and activity for recombinant S. aureus dnaN?

For purifying recombinant S. aureus dnaN, a multi-step purification strategy similar to that used for other S. aureus proteins is recommended. Based on methodologies for similar proteins, consider the following approach:

• Initial capture using affinity chromatography (His-tag or GST-tag depending on construct design)

• Intermediate purification via ion exchange chromatography to separate charged variants

• Final polishing step using size exclusion chromatography to ensure homogeneity and remove aggregates

For antibody proteins expressed in HEK293F cells, affinity chromatography with protein A or G columns followed by buffer exchange has yielded preparations with high purity . Similar principles could apply to dnaN, though specific optimization would be required. Critical parameters include buffer composition, pH maintenance, and addition of stabilizing agents such as glycerol to preserve activity during storage.

How can I develop an assay to measure the DNA-binding affinity of recombinant dnaN?

To quantitatively evaluate the DNA-binding properties of recombinant S. aureus dnaN, several complementary approaches can be implemented:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified dnaN with fluorescently-labeled DNA oligonucleotides

  • Analyze complex formation by native PAGE

  • Calculate apparent Kd values from bound versus unbound DNA fractions

• Surface Plasmon Resonance (SPR):

  • Immobilize biotinylated DNA on a streptavidin sensor chip

  • Flow recombinant dnaN protein at various concentrations

  • Determine kon and koff rates to calculate binding affinity

• Microscale Thermophoresis (MST):

  • Label either DNA or protein with a fluorescent dye

  • Measure changes in thermophoretic mobility upon binding

  • Derive binding constants from concentration-dependent changes

Drawing from ELISA methodologies used for S. aureus antibodies , similar principles of dose-response curve analysis can be applied. The EC50 value can be calculated from curve fitting to a four-parameter logistic equation, and the limit of detection (LOD) calculated based on the equation: Y = bottom + (top – bottom)/(1 + 10((log EC50 – X) × HillSlope)) .

How does the sequence conservation of dnaN across Staphylococcal species impact structural studies and inhibitor design?

The evolutionary conservation of DNA polymerase III components across bacterial species has important implications for structural studies and antimicrobial development. Analysis of nearly 2000 bacterial genomes revealed that all encode one or more DNA polymerase III α-subunit homologs, suggesting essential functions preserved through evolution . For the beta subunit (dnaN), similar conservation patterns likely exist.

Conservation analysis should consider:

• Core structural elements required for DNA binding

• Interface residues for polymerase interaction

• Species-specific variations that could affect inhibitor specificity

This information can guide structure-based drug design efforts targeting the sliding clamp. Key methodological approaches include:

• Multiple sequence alignment of dnaN sequences from diverse Staphylococcal species

• Homology modeling based on available crystal structures

• Molecular dynamics simulations to identify conserved functional motions

• Virtual screening against conserved binding pockets

What are the protein-protein interaction profiles of S. aureus dnaN with other replication and repair proteins?

The beta sliding clamp serves as a hub for multiple protein-protein interactions in bacterial replication. To characterize the interaction network of S. aureus dnaN:

• Yeast two-hybrid screening with dnaN as bait against a library of S. aureus proteins

• Co-immunoprecipitation studies followed by mass spectrometry

• Surface plasmon resonance with purified potential partner proteins

• Crosslinking mass spectrometry to map interaction interfaces

Comparative analysis with other bacterial systems suggests dnaN likely interacts with:

• DNA polymerase III alpha, epsilon, and delta subunits

• DNA repair polymerases (IV and V)

• Ligase and mismatch repair proteins

• Cell cycle regulation factors

These interactions collectively contribute to replication coordination and DNA damage tolerance, with potentially unique features in S. aureus compared to model organisms like E. coli.

How can the solubility of recombinant S. aureus dnaN be improved during heterologous expression?

Producing soluble recombinant dnaN can be challenging due to protein misfolding or aggregation. Several strategies can address this issue:

• Fusion tags optimization:

  • Test multiple solubility-enhancing tags (MBP, SUMO, TrxA)

  • Optimize tag placement (N-terminal vs. C-terminal)

  • Include flexible linkers between tag and protein

• Expression conditions modification:

  • Reduce induction temperature (16-20°C)

  • Decrease inducer concentration

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

• Lysis buffer optimization:

  • Include stabilizing agents (glycerol, arginine)

  • Test various detergents at sub-CMC concentrations

  • Adjust salt concentration and pH

When using mammalian expression systems like HEK293F, optimizing cell density (3×10⁵-3×10⁶ cells/mL) before protein expression and maintaining proper incubation conditions (37°C, 8.0% CO₂) can significantly improve protein yield and solubility .

What methods can detect conformational changes in dnaN during different stages of the replication cycle?

To monitor conformational dynamics of the dnaN sliding clamp during replication:

• FRET-based approaches:

  • Site-specific labeling of recombinant dnaN with fluorophore pairs

  • Monitor distance changes between domains upon DNA binding or protein partner interaction

  • Perform measurements in solution or at single-molecule level

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare deuterium uptake patterns in different functional states

  • Identify regions with altered solvent accessibility

  • Map structural changes to specific domains

• Cryo-electron microscopy:

  • Capture dnaN in complexes representing different functional states

  • Perform 3D reconstruction to visualize conformational changes

  • Correlate structural changes with biochemical activities

These approaches can reveal mechanisms of clamp loading, polymerase exchange, and other dynamic processes that are difficult to capture with static structural methods.

How does S. aureus dnaN differ from E. coli dnaN in structure and function?

Comparative analysis of DNA polymerase III components across bacterial species reveals both conservation and divergence. While the search results focus on the alpha subunit, similar principles apply to the beta subunit:

These differences may reflect adaptations to different cellular environments and replication requirements. Methodological approaches to explore these differences include:

• Structural comparison through X-ray crystallography or cryo-EM

• Cross-species complementation experiments

• Chimeric protein construction and functional testing

• Comparative biochemical assays under varied conditions

What emerging technologies are advancing our understanding of DNA replication machinery in S. aureus?

Several cutting-edge approaches are transforming research on bacterial replication proteins like dnaN:

• CRISPR-Cas9 genome editing:

  • Precise modification of chromosomal dnaN

  • Creation of conditional mutants for essential function studies

  • Introduction of tagged versions for in vivo imaging

• Single-molecule techniques:

  • Real-time visualization of replisome assembly and function

  • Direct measurement of sliding clamp dynamics on DNA

  • Quantification of force generation and mechanical properties

• Integrative structural biology:

  • Combining cryo-EM, X-ray crystallography, and computational modeling

  • Building comprehensive models of the replisome architecture

  • Simulating replication dynamics at atomic resolution

• Systems biology approaches:

  • Network analysis of replication protein interactions

  • Multi-omics studies of replication stress responses

  • Mathematical modeling of replication timing and coordination

These technologies promise to reveal new mechanistic insights into S. aureus DNA replication that could inform novel antimicrobial strategies targeting the replication machinery.