Recombinant Synechocystis sp. DNA primase (dnaG), partial

What is the structural organization of DNA primase (dnaG) in Synechocystis sp. PCC 6803?

Synechocystis sp. DNA primase (dnaG) belongs to the topoisomerase-primase (Toprim) domain superfamily. The protein contains several conserved structural elements that define its functional architecture. The Toprim domain encompasses two critical motifs: motif IV featuring an invariant glutamate, and motif V characterized by an aspartate dyad (DxD) preceded by conserved hydrophobic regions predicted to form β-strands . These conserved motifs are directly involved in the catalytic function of primer synthesis.

Structurally, bacterial DnaG primases like those in Synechocystis contain a distinctive N-terminal Zn-chelating domain that facilitates DNA binding and confers weak sequence specificity for primer initiation sites. Additionally, the C-terminal domain interacts with the DnaB helicase, enabling coordination during DNA replication . This multi-domain architecture allows DNA primase to perform its essential role in DNA replication.

How does the function of DNA primase in Synechocystis differ from that in other organisms?

Unlike many bacteria with a single DnaG-type primase, genomic analyses reveal that Synechocystis contains multiple primase-like genes. This complexity is particularly evident when comparing Synechocystis to model organisms like E. coli. Moreover, archaeal organisms possess DnaG homologs that show greater similarity to bacterial DnaG but are likely involved in DNA repair rather than replication, given the presence of eukaryotic-type primase subunits in archaea . This suggests specialized or diversified functions of the primase variants in Synechocystis that may relate to its photosynthetic lifestyle and unique genomic organization with multiple chromosome copies.

The conservation pattern of the Toprim domain across these diverse organisms indicates an ancient evolutionary origin of this catalytic module , with Synechocystis representing an interesting evolutionary position between bacteria and plants.

What are the most effective methods for expressing recombinant Synechocystis dnaG in heterologous systems?

For successful expression of recombinant Synechocystis dnaG in heterologous systems, researchers should consider the following methodological approaches:

• Vector Selection: Choose expression vectors with promoters that function effectively in the host organism. For E. coli expression systems, T7-based expression vectors are commonly used for recombinant cyanobacterial proteins.

• Codon Optimization: The GC-rich codon usage of Synechocystis (approximately 47.7%) can cause expression difficulties in heterologous hosts. Codon optimization for the expression host can significantly improve protein yield.

• Purification Strategy: Adding affinity tags (His-tag, GST, etc.) facilitates purification. The optimal position of the tag (N- or C-terminal) should be determined experimentally to avoid interfering with protein folding or activity.

• Expression Conditions: Lower temperatures (16-20°C) often improve the solubility of cyanobacterial proteins in E. coli. Additionally, consider including specific cofactors or metal ions required for proper folding.

For native expression, integration of the gene into the Synechocystis genome can be verified using PCR analysis with primers binding to different regions of the locus. Genomic DNA from wild-type strains serves as a control . Expression can be confirmed through RT-PCR using gene-specific primers with appropriate controls (samples lacking RT or containing water instead of template) .

How can I verify successful integration and expression of recombinant dnaG in Synechocystis?

Verification of successful integration and expression of recombinant dnaG in Synechocystis requires a multi-step approach:

• PCR Verification of Integration: Design multiple primer pairs that bind to different regions of the integrated construct and adjacent genomic regions. This strategy allows confirmation of proper insertion at the target locus. For example, using primers that span the junction between the native genome and the inserted gene will produce amplification products of specific sizes only if integration occurred at the correct site .

• Segregation Analysis: Synechocystis possesses multiple genome copies, necessitating verification that all copies contain the integrated gene (complete segregation). This can be achieved through PCR analysis using primers that would produce different-sized products from wild-type and transformed genomes .

• Transcription Analysis: RT-PCR using gene-specific primers confirms transcription of the integrated gene. Include controls such as samples without reverse transcriptase and water instead of template to rule out DNA contamination and non-specific amplification .

• Southern Blot Analysis: For definitive confirmation of integration and complete segregation, Southern blot analysis provides robust verification of the genomic structure .

• Protein Expression Verification: Western blot analysis using antibodies specific to the recombinant protein or to an included tag (e.g., His-tag) can confirm expression at the protein level.

The following table summarizes the verification methods and expected outcomes:

How does environmental stress impact dnaG expression and function in Synechocystis sp. PCC 6803?

Environmental stress significantly modulates dnaG expression and function in Synechocystis sp. PCC 6803, with distinct patterns emerging under different stress conditions. Transcriptomic analyses reveal that Synechocystis responds to environmental stimuli through sophisticated transcriptional rewiring .

Under nutrient depletion conditions (carbon, nitrogen, phosphate), expression patterns of genes involved in DNA replication, including dnaG, undergo significant changes. These changes are part of a broader stress response that affects thousands of transcriptional units across the genome . Similarly, iron stress induces specific transcriptional responses that may impact DNA replication machinery.

The stress response in Synechocystis is coordinated through complex regulatory networks that include small RNAs (sRNAs) with condition-specific expression patterns. Several sRNAs have been identified with functions specific to different stress conditions: carbon depletion (CsiR1), nitrogen depletion (NsiR4), phosphate depletion (PsiR1), iron stress (IsaR1), and photosynthesis (PsrR1) . These sRNAs may directly or indirectly regulate dnaG expression under specific stress conditions.

The integration of transcriptomic data with functional analyses provides a comprehensive understanding of how dnaG responds to environmental perturbations, offering insights for engineering more resilient strains for biotechnological applications.

What are the implications of mutations in the conserved Toprim domain of Synechocystis dnaG?

Mutations in the conserved Toprim domain of Synechocystis dnaG can have profound implications for DNA replication, cell viability, and genome stability. The Toprim domain contains critical catalytic residues essential for primase function, particularly the invariant glutamate in motif IV and the DxD motif in motif V .

Site-directed mutagenesis studies in related systems indicate that alterations to these conserved residues drastically reduce or abolish primase activity. The invariant glutamate likely participates in catalysis through coordination of metal ions required for the nucleotidyl transfer reaction, while the DxD motif is involved in binding divalent metal ions necessary for catalytic activity .

The high conservation of these motifs across diverse organisms from bacteria to archaea underscores their functional significance. Mutational studies can be designed based on known mutations in related organisms, as demonstrated by the successful creation of free-running period (FRP) mutants in Synechocystis based on mutations identified in S. elongatus .

Researchers investigating Toprim domain mutations should consider:

• The effects on primase catalytic activity through in vitro assays

• Impacts on cell growth and viability

• Potential compensatory mechanisms through other primase-like proteins in Synechocystis

• Structural changes using crystallographic or molecular dynamics approaches

• Interactions with other replication machinery components

The development of mutation strategies based on known mutations in related systems provides a powerful approach for investigating the functional significance of specific residues in the Toprim domain .

What are the optimal conditions for isolating and purifying recombinant Synechocystis dnaG protein?

Optimal isolation and purification of recombinant Synechocystis dnaG protein requires careful consideration of buffer conditions, purification techniques, and protein stability factors. The following methodological approach ensures high yield and activity:

• Cell Lysis Buffer Optimization:

  • 50 mM Tris-HCl (pH 7.5-8.0)

  • 300-500 mM NaCl (to maintain solubility)

  • 5-10% glycerol (stabilizes protein structure)

  • 1-5 mM β-mercaptoethanol or DTT (maintains reduced state)

  • 0.1-0.5 mM EDTA (chelates metal ions that might promote oxidation)

  • Protease inhibitor cocktail (prevents degradation)

• Affinity Chromatography:
  For His-tagged recombinant dnaG:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Binding buffer: lysis buffer with 10-20 mM imidazole

  • Wash buffer: lysis buffer with 20-50 mM imidazole

  • Elution buffer: lysis buffer with 250-300 mM imidazole

• Secondary Purification:

  • Ion exchange chromatography (theoretical pI of dnaG should guide selection)

  • Size exclusion chromatography (final polishing step and buffer exchange)

• Activity Preservation:

  • Include divalent cations (Mg²⁺ or Mn²⁺) at 1-5 mM in storage buffers

  • The Toprim domain requires these cations for catalytic activity

  • Store with 10-20% glycerol at -80°C in small aliquots

• Quality Control:

  • SDS-PAGE for purity assessment

  • Western blot for identity confirmation

  • Dynamic light scattering for aggregation analysis

  • Circular dichroism for secondary structure verification

The purification protocol may require optimization based on specific recombinant constructs and expression systems. Monitoring enzyme activity throughout purification helps identify steps that may compromise protein function.

How can I design PCR primers for accurate amplification and mutagenesis of Synechocystis dnaG?

Designing effective PCR primers for amplification and mutagenesis of Synechocystis dnaG requires consideration of several critical factors:

• Genome Context Analysis:

  • Obtain the complete genomic sequence around the dnaG gene

  • Analyze flanking regions for unique sequences suitable for primer design

  • Consider the high GC content typical of Synechocystis DNA

• Primer Design for Amplification:

  • Optimal primer length: 18-30 nucleotides

  • GC content: 40-60% (balanced within the primer)

  • Melting temperature (Tm): 55-65°C, with paired primers within 5°C of each other

  • Avoid secondary structures (hairpins, self-dimers)

  • Include restriction sites with 3-6 nucleotide overhangs if needed for cloning

  • For verification of gene integration, design primer pairs that bind to different regions of the target locus and flanking sequences

• Primer Design for Site-Directed Mutagenesis:

  • Center the mutation in the primer with 10-15 perfectly matched nucleotides on either side

  • For mutations in the conserved Toprim domain, target the invariant glutamate in motif IV or the DxD motif in motif V

  • Consider known mutations from related organisms like S. elongatus that affect protein function

  • Use complementary primer pairs with the same mutation

  • Keep primer length between 25-45 nucleotides

• PCR Optimization for Synechocystis DNA:

  • Include DMSO (5-10%) or betaine (1-2 M) to reduce secondary structure formation in GC-rich regions

  • Use touchdown PCR protocols to improve specificity

  • Consider hot-start polymerases to reduce non-specific amplification

  • Test gradient PCR to identify optimal annealing temperatures

• Verification Strategies:

  • Design sequencing primers that allow complete coverage of the amplified region

  • Include primers for RT-PCR to verify transcription of the integrated gene

  • Design primer pairs that distinguish between wild-type and mutant alleles for segregation analysis

The table below presents examples of primer design approaches for different applications:

How do I troubleshoot common issues encountered during recombinant dnaG expression and purification?

Recombinant dnaG expression and purification can present several challenges. The following troubleshooting guide addresses common issues with methodological solutions:

• Low Expression Levels:

  • Issue: Minimal production of recombinant dnaG protein.

  • Solutions:

    • Optimize codon usage for the expression host

    • Test different promoter systems (T7, tac, arabinose-inducible)

    • Reduce expression temperature (16-20°C)

    • Adjust inducer concentration and induction timing

    • Consider fusion partners (MBP, SUMO) to enhance solubility

• Protein Insolubility:

  • Issue: Formation of inclusion bodies containing inactive protein.

  • Solutions:

    • Express at lower temperatures with reduced inducer concentration

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

    • Add solubility-enhancing agents to lysis buffer (0.1% Triton X-100, 500 mM arginine)

    • Consider refolding protocols if inclusion bodies persist

• Proteolytic Degradation:

  • Issue: Fragmented protein bands observed during purification.

  • Solutions:

    • Use protease-deficient expression strains

    • Include multiple protease inhibitors in all buffers

    • Reduce handling time and maintain samples at 4°C

    • Consider repositioning affinity tags if cleavage occurs at tag junctions

• Poor Affinity Binding:

  • Issue: Target protein flows through during affinity chromatography.

  • Solutions:

    • Verify tag is in-frame and not occluded in protein structure

    • Optimize binding conditions (buffer pH, salt concentration)

    • Increase imidazole in binding buffer to reduce non-specific binding

    • Consider alternative tag placement (N vs. C-terminal)

• Loss of Enzymatic Activity:

  • Issue: Purified protein lacks primase activity.

  • Solutions:

    • Add divalent cations (Mg²⁺/Mn²⁺) required for Toprim domain function

    • Include reducing agents to maintain active site cysteines

    • Verify protein folding using circular dichroism

    • Consider co-purification with interacting partners

When analyzing expression in Synechocystis, verify complete segregation of all genome copies. PCR analysis should confirm that all copies contain the integrated gene and not the wild-type allele . For gene expression verification, include comprehensive controls in RT-PCR experiments to distinguish genuine transcription from potential DNA contamination .

What approaches can resolve data inconsistencies when comparing dnaG activity across different experimental conditions?

Resolving data inconsistencies when comparing dnaG activity across different experimental conditions requires systematic analysis of potential variables and implementation of standardization protocols:

• Standardization of Enzyme Activity Assays:

  • Define a standard unit of primase activity

  • Ensure consistent reaction conditions (temperature, pH, ionic strength)

  • Use internal controls with known activity in each assay

  • Validate activity measurements using multiple independent methods

• Comprehensive Controls:

  • Include positive controls (commercially available or well-characterized primases)

  • Use negative controls (heat-inactivated enzyme, reaction mixtures missing essential components)

  • Include technical replicates (same sample measured multiple times)

  • Incorporate biological replicates (independent protein preparations)

• Identification of Confounding Variables:

  • Analyze buffer composition effects on activity (salt type/concentration, pH)

  • Evaluate the impact of post-translational modifications

  • Assess effects of different affinity tags on enzyme function

  • Consider batch-to-batch variation in reagents

• Statistical Analysis for Reconciling Contradictory Data:

  • Apply appropriate statistical methods to determine significance of differences

  • Consider meta-analysis approaches for integrating multiple datasets

  • Use Bland-Altman plots to identify systematic biases between methods

  • Calculate coefficients of variation to assess reproducibility

• Addressing Environmental Sensitivity:

  • Recognize that dnaG expression in Synechocystis responds to environmental stimuli

  • Document all environmental parameters during experiments

  • Design specific experiments to test hypotheses about environmental effects

  • Consider circadian regulation effects, as Synechocystis has complex clock gene organization

When analyzing transcriptomic data, ensure proper normalization methods are applied, especially when comparing across different environmental conditions. The transcriptional landscape of Synechocystis undergoes significant reorganization under different growth conditions , which may affect interpretation of dnaG activity data.

What are promising approaches for studying the interaction between Synechocystis dnaG and other components of the DNA replication machinery?

Several promising approaches can elucidate the interactions between Synechocystis dnaG and other components of the DNA replication machinery:

• Protein-Protein Interaction Studies:

  • Bacterial two-hybrid systems adapted for cyanobacterial proteins

  • Co-immunoprecipitation followed by mass spectrometry

  • Surface plasmon resonance to determine binding kinetics

  • Fluorescence resonance energy transfer (FRET) for in vivo interaction analysis

  • Native mass spectrometry to characterize multiprotein complexes

• Structural Biology Approaches:

  • X-ray crystallography of dnaG alone and in complexes

  • Cryo-electron microscopy of larger replication assemblies

  • NMR spectroscopy for dynamic interaction studies

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Integrative structural modeling combining multiple experimental data types

• Functional Genomics:

  • CRISPR interference for controlled downregulation of interacting partners

  • Synthetic genetic array analysis to identify genetic interactions

  • Global protein-DNA interaction mapping using ChIP-seq

  • Transcriptome profiling under different growth conditions to identify co-regulated genes

  • Whole-genome sequencing of suppressor mutants

• Advanced Microscopy:

  • Super-resolution microscopy to visualize replication complexes

  • Single-molecule tracking to follow dynamics of tagged replication proteins

  • Microfluidics combined with time-lapse microscopy for real-time analysis

  • Expansion microscopy for improved spatial resolution in bacterial cells

The C-terminal domain of bacterial DnaG interacts with the DnaB helicase , making this a primary target for interaction studies. Additionally, the presence of multiple primase-like proteins in Synechocystis suggests potential for specialized interactions or redundancy that warrants investigation.

How might CRISPR-Cas technologies be applied to study and modify dnaG function in Synechocystis?

CRISPR-Cas technologies offer powerful approaches for studying and modifying dnaG function in Synechocystis:

• Gene Editing Applications:

  • Precise modification of conserved Toprim domain residues to study structure-function relationships

  • Creation of domain swaps between different primase-like genes in Synechocystis

  • Introduction of mutations known to affect primase function in related organisms

  • Generation of conditional knockdowns or temperature-sensitive alleles

  • Engineering fusion proteins with fluorescent tags for localization studies

• CRISPR Interference (CRISPRi) Applications:

  • Tunable repression of dnaG expression to determine minimal functional levels

  • Simultaneous repression of multiple primase-like genes to identify redundancy

  • Temporal control of dnaG expression to study replication timing

  • Targeting upstream regulatory elements to dissect transcriptional control

  • Creation of synthetic genetic interaction maps with other DNA replication genes

• Methodological Considerations for Synechocystis:

  • Optimize Cas9 expression for the high-GC content of Synechocystis

  • Design sgRNAs with specificity verification against the entire genome

  • Incorporate selectable markers for enrichment of edited cells

  • Consider the polyploid nature of Synechocystis when designing segregation strategies

  • Verify homozygosity of edits across all genome copies using methods similar to those used for gene integration

• Advanced CRISPR Applications:

  • Prime editing for precise base changes without double-strand breaks

  • CRISPR activation (CRISPRa) to upregulate dnaG in specific conditions

  • Multiplex genome editing to simultaneously modify multiple replication genes

  • Saturation mutagenesis of key Toprim domain regions

  • Development of CRISPR-based biosensors for monitoring replication stress

The polyploid nature of Synechocystis requires careful design of CRISPR-based experiments, particularly in ensuring complete segregation of mutations across all genome copies. PCR verification strategies similar to those used for gene integration can confirm that all copies contain the desired edit .

How does Synechocystis dnaG differ structurally and functionally from E. coli dnaG and other bacterial primases?

Synechocystis dnaG shares fundamental structural elements with other bacterial primases while exhibiting distinct characteristics that reflect its cyanobacterial origin:

• Domain Organization Comparison:

  • Shared Features: Like E. coli dnaG, Synechocystis primase contains the three canonical domains: an N-terminal zinc-binding domain, a central RNA polymerase domain (the Toprim domain), and a C-terminal helicase-binding domain .

  • Distinctive Elements: Synechocystis possesses multiple primase-like genes, suggesting functional specialization not observed in E. coli.

• Conserved Catalytic Motifs:

  • High Conservation: The critical motifs IV and V in the Toprim domain, containing the invariant glutamate and DxD motif, are highly conserved between Synechocystis and other bacterial primases .

  • Functional Implication: This conservation suggests a shared catalytic mechanism across bacterial primases despite divergent evolutionary histories.

• Phylogenetic Position:

  • Evolutionary Context: Bacterial DnaG primases form a monophyletic assemblage with distinctive structural features .

  • Cyanobacterial Specificity: Synechocystis dnaG represents a specific cyanobacterial adaptation within this broader evolutionary context.

• Multiple Primase-Like Proteins:

  • Unique Feature: Unlike E. coli with a single DnaG primase, Synechocystis contains multiple primase-like genes.

  • Functional Divergence: This suggests potential specialization or redundancy not observed in model organisms like E. coli.

• Environmental Responsiveness:

  • Differential Regulation: Transcriptomic analyses reveal that Synechocystis dnaG expression responds to environmental conditions , potentially reflecting adaptation to photosynthetic lifestyle.

  • Stress Response: The complex transcriptional landscape of Synechocystis shows condition-specific responses not characterized in the same detail for E. coli dnaG.

The table below summarizes key differences between Synechocystis dnaG and E. coli dnaG:

What insights from archaeal primases can be applied to understanding Synechocystis dnaG function?

Insights from archaeal primases provide valuable perspectives for understanding Synechocystis dnaG function, particularly given the evolutionary relationships within the Toprim domain superfamily:

• Evolutionary Relationships:

  • Archaeal organisms possess DnaG homologs that show similarity to bacterial DnaG proteins in the Toprim domain .

  • These archaeal DnaG-like proteins likely function in DNA repair rather than replication, as archaea typically utilize eukaryotic-type primase subunits for replication .

  • This functional divergence provides a framework for investigating potential non-replicative roles of some Synechocystis primase-like proteins.

• Domain Architecture Insights:

  • Archaeal DnaG-like proteins contain an N-terminal domain with a conserved motif similar to motif VI of superfamily II helicases .

  • This domain may function in nucleic acid binding, similar to the Zn-binding domain in bacterial DnaG.

  • Comparative analysis of these domains could reveal functional adaptations in Synechocystis dnaG.

• Methodological Applications:

  • Structural studies of archaeal primases have employed techniques that could be applied to Synechocystis dnaG.

  • Archaeal primase biochemical assays provide templates for developing Synechocystis-specific activity assays.

  • Heterologous expression strategies optimized for archaeal proteins might be adapted for Synechocystis proteins.

• Structure-Function Relationships:

  • Analysis of conserved catalytic residues between archaeal and bacterial primases helps identify the most critical amino acids for function.

  • The conservation of the Toprim domain across diverse organisms underscores its fundamental importance in nucleic acid metabolism .

  • Mutations that affect archaeal primase function might guide targeted mutagenesis studies in Synechocystis dnaG.

• Non-Canonical Functions:

  • Archaeal DnaG homologs likely function in DNA repair rather than replication .

  • This suggests the possibility that some Synechocystis primase-like proteins might have specialized roles beyond conventional primer synthesis.

  • Investigation of potential repair functions would represent a novel research direction for Synechocystis dnaG studies.

The study of archaeal primases provides a broader evolutionary context for understanding the functional diversity of the Toprim domain superfamily, offering insights that can guide research on specialized functions of Synechocystis dnaG and related proteins.