Recombinant Takifugu rubripes Structural maintenance of chromosomes protein 5 (smc5), partial

What is the Structural Maintenance of Chromosomes protein 5 (SMC5) in Takifugu rubripes and why is it important in genomic research?

SMC5 is a critical component of the Smc5/6 complex, one of three essential SMC complexes (along with cohesin and condensin) involved in higher-order chromosome structure organization. This protein plays fundamental roles in genome stability, DNA repair, and chromosome segregation. In Takifugu rubripes (Japanese pufferfish), studying SMC5 is particularly valuable because:

• Fugu has a compact genome (~384 Mb) with similar gene content to humans but with reduced intergenic regions, making it an excellent comparative genomics model

• The SMC5/6 complex in fugu forms distinctive toroidal structures with unique subunit interfaces (molecular latch and functional hub) that are absent in other SMC complexes

• Mutations in these unique interfaces cause severe phenotypic effects, including sensitivity to DNA-damaging agents, demonstrating their functional importance

The recombinant partial SMC5 protein serves as a valuable tool for investigating these mechanisms in controlled experimental settings.

How does the SMC5 protein in Takifugu rubripes compare to homologs in other vertebrate species?

The SMC5 protein is highly conserved across vertebrates, reflecting its essential cellular functions. Key comparative aspects include:

• Takifugu rubripes SMC5 shows considerable sequence conservation with homologs in other vertebrates, including humans, Xenopus laevis, and Saccharomyces cerevisiae

• Despite conservation, the Smc5/6 complex in fugu contains distinctive subunit interfaces not found in other organisms, suggesting species-specific functional adaptations

• The fugu genome's compact nature (approximately 1/8 the size of the human genome) makes it particularly useful for identifying conserved functional domains and regulatory elements in SMC5

Table 1: Comparison of SMC5 across select species

What are the optimal expression systems for producing functional recombinant Takifugu rubripes SMC5 protein?

Several expression systems have been utilized for SMC5 production, each with advantages and limitations:

• E. coli expression: Most commonly used for partial domains of SMC5. While providing high yields, E. coli often struggles with proper folding of full-length SMC proteins due to their size and complexity

• Baculovirus expression: Provides improved folding and post-translational modifications for larger SMC protein fragments, though with lower yields than bacterial systems

• Yeast expression systems: Offer a eukaryotic environment more suitable for functional studies of SMC5, particularly when investigating interactions with other SMC components

For optimal results, specific methodology should include:

• Codon optimization for the selected expression system

• Addition of solubility-enhancing tags (His, GST, or MBP)

• Expression at reduced temperatures (16-18°C for E. coli)

• Use of specialized strains designed for expression of challenging proteins

What purification strategies are most effective for obtaining highly pure recombinant Takifugu rubripes SMC5 protein?

Effective purification typically employs a multi-step approach:

• Initial capture: Affinity chromatography using nickel-NTA for His-tagged constructs achieves >85% purity as demonstrated in commercial preparations

• Intermediate purification: Ion-exchange chromatography to separate charge variants

• Polishing: Size-exclusion chromatography to eliminate aggregates and achieve final purity >95%

• Quality control: SDS-PAGE and Western blotting to confirm identity and purity

For structural studies requiring exceptionally pure protein:

• Tag removal using specific proteases (TEV, PreScission)

• Additional chromatography steps to remove cleaved tags

• Dynamic light scattering to verify monodispersity

How does recombinant Takifugu rubripes SMC5 interact with DNA, and what experimental approaches best characterize these interactions?

The Smc5/6 complex shows preferential binding to single-stranded DNA (ssDNA), and this interaction is mediated through its distinctive hinge domain architecture. Key experimental approaches include:

• Electrophoretic mobility shift assays (EMSAs): Demonstrate the preference of the Smc5/6-hinge complex for ssDNA over double-stranded DNA

• Surface plasmon resonance (SPR): Quantifies binding kinetics and affinity constants for SMC5 interaction with various DNA structures

• DNA pull-down assays: Identify specific DNA structures preferentially bound by SMC5

• Fluorescence anisotropy: Measures real-time binding dynamics with labeled DNA substrates

Research has shown that mutations in the unique "latch" and "hub" interfaces of the Smc5/6 hinge significantly affect DNA binding properties, suggesting these regions play critical roles in controlling DNA association .

What genomic resources and tools are available for studying Takifugu rubripes smc5 gene and its protein product?

Several valuable genomic resources exist:

• Genome assemblies: The latest fTakRub1.2 assembly from 2019 provides chromosome-level organization with 22 chromosomes assembled, scaffold N50 of 16,705,553, and contig N50 of 3,136,617

• Gene annotation database: Complete annotation available through Ensembl, including transcript variants and protein domains

• Recombinant proteins: Commercial sources provide partial recombinant Takifugu rubripes SMC5 protein with >85% purity

• Genetic linkage maps: Available genetic maps with microsatellite markers can help place smc5 in the broader genomic context

Table 2: Key Genomic Resources for Takifugu rubripes SMC5 Research

How can recombinant Takifugu rubripes SMC5 be utilized in structural biology studies to understand the molecular mechanisms of chromosome maintenance?

Recombinant Takifugu rubripes SMC5 offers several advantages for structural biology:

• The compact nature of fugu proteins often results in more stable recombinant constructs suitable for crystallization

• The unique "latch" and "hub" interfaces of the Smc5/6 hinge domain provide excellent targets for structure-function studies

• Methodological approaches include:

  • X-ray crystallography of isolated domains, particularly the hinge region

  • Cryo-electron microscopy for full complex visualization

  • Small-angle X-ray scattering (SAXS) for solution structure determination

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

Researchers should consider expressing both wild-type and mutant versions of the protein to investigate how specific amino acid changes affect structure and function.

What are the challenges in reconstituting a functional Smc5/6 complex using recombinant Takifugu rubripes components?

Reconstituting the complete Smc5/6 complex presents several challenges:

• The complex contains multiple subunits beyond SMC5, including SMC6 and several non-SMC elements (Nse1-6)

• Correct stoichiometry is critical for functional activity

• The complex undergoes ATP-dependent conformational changes essential for function

Successful reconstitution strategies include:

• Co-expression of multiple subunits in a eukaryotic system

• Sequential assembly with purified components under controlled conditions

• Verification of complex integrity through analytical techniques (analytical ultracentrifugation, native gel electrophoresis)

• Functional validation through DNA binding and ATPase activity assays

Recent studies have shown that the distinctive "latch" and "hub" interfaces in the Smc5/6 hinge domain are critical for complex stability and function, making these regions particularly important to preserve during reconstitution .

How has the SMC5 protein evolved in the Takifugu lineage compared to other teleost fish and vertebrates?

Evolutionary analysis reveals:

• The pufferfish lineage, including Takifugu rubripes, has undergone explosive speciation in East Asian marine environments, with approximately 25 Takifugu species identified

• Despite the compact genome of Takifugu (approximately 1/8 the size of the human genome), the number of protein-coding genes (including smc5) is largely conserved with other vertebrates

• The unique structural features in the Smc5/6 hinge domain suggest lineage-specific adaptations that may reflect specialized functions in genome maintenance

Comparative genomic approaches can identify:

• Conserved functional domains indicating essential activities

• Lineage-specific sequence variations that might confer specialized functions

• Evolutionary rates of different protein domains, with functional cores typically showing higher conservation

What are the most common challenges in working with recombinant Takifugu rubripes SMC5, and how can they be overcome?

Common challenges include:

• Protein solubility issues: SMC proteins are large and prone to aggregation

  • Solution: Use solubility-enhancing tags (MBP, SUMO), optimize buffer conditions, express at lower temperatures (16-18°C)

• Protein stability concerns: Partial degradation during purification

  • Solution: Include protease inhibitors, minimize freeze-thaw cycles, store at appropriate concentration with glycerol (recommended 5-50% final concentration)

• Expression yield limitations: Low yields in certain expression systems

  • Solution: Optimize codon usage, consider different expression systems (bacterial, insect, yeast)

• Functional activity assessment: Challenges in demonstrating native activity

  • Solution: Design appropriate DNA binding assays, include positive controls, ensure proper protein folding

Commercial suppliers recommend avoiding repeated freeze-thaw cycles and suggest storing working aliquots at 4°C for up to one week .

What experimental controls are essential when studying the DNA binding properties of recombinant Takifugu rubripes SMC5?

Essential controls include:

• Negative controls:

  • Heat-denatured protein to demonstrate specificity of binding

  • Irrelevant proteins of similar size and charge properties

  • Buffer-only conditions to establish baseline

• Positive controls:

  • Well-characterized SMC proteins from other organisms with known binding properties

  • Other domains of the Smc5/6 complex with established DNA interactions

• Substrate controls:

  • Various DNA structures (ssDNA, dsDNA, branched structures)

  • Different lengths and sequences to determine specificity

  • Labeled and unlabeled competitors to confirm specificity

• Biochemical validation:

  • ATPase activity measurements to confirm functional integrity

  • Conformational analysis using circular dichroism or thermal shift assays

Research has shown that the Smc5/6-hinge complex binds preferentially to ssDNA, and mutations in the unique "latch" and "hub" interfaces significantly affect this interaction , making wild-type versus mutant comparisons particularly informative.

What emerging techniques might advance our understanding of Takifugu rubripes SMC5 function in genome maintenance?

Several cutting-edge approaches show promise:

• CRISPR-Cas9 genome editing in Takifugu cell lines to:

  • Create specific mutations in the unique latch and hub domains

  • Generate fluorescently tagged endogenous SMC5 for live imaging

  • Evaluate phenotypic consequences of SMC5 dysregulation

• Single-molecule approaches:

  • Optical tweezers to study SMC5-mediated DNA compaction

  • DNA curtains to visualize protein-DNA interactions in real-time

  • FRET-based assays to detect conformational changes

• Integrative omics approaches:

  • ChIP-seq to map genome-wide binding sites

  • Hi-C to assess effects on 3D genome organization

  • Proteomics to identify interaction partners in different cellular contexts

• Cryo-electron tomography:

  • Visualization of SMC5 in its native cellular environment

  • Structural characterization of the complete Smc5/6 complex

How might studies of Takifugu rubripes SMC5 contribute to our understanding of human disease mechanisms?

Research on Takifugu rubripes SMC5 has several potential implications for human health:

• Cancer biology:

  • SMC5/6 complex dysfunction is associated with genome instability, a hallmark of cancer

  • Understanding the molecular mechanisms of SMC5 function could identify novel therapeutic targets

• Developmental disorders:

  • Mutations in SMC complex components cause several human developmental syndromes

  • The compact genome of fugu facilitates identification of critical functional domains and regulatory elements

• Aging and senescence:

  • SMC5/6 plays roles in managing replication stress and telomere maintenance, processes linked to aging

  • Comparative studies between short-lived and long-lived organisms may reveal evolutionary adaptations

• DNA repair deficiency syndromes:

  • SMC5/6 complex is crucial for homologous recombination repair

  • Characterizing specific functions may help understand human disorders with defects in this pathway