Recombinant Salmo salar DNA repair protein SWI5 homolog (swi5)

What is the SWI5 homolog in Atlantic salmon (Salmo salar)?

The SWI5 homolog in Atlantic salmon is a DNA repair protein involved in homologous recombination processes. Based on comparative studies with other species, Salmo salar SWI5 likely forms a complex with MEI5, similar to the human SWI5-MEI5 complex that facilitates DNA repair . The Atlantic salmon genome underwent a salmonid-specific fourth vertebrate whole-genome duplication (Ss4R) approximately 80 million years ago , which may have resulted in duplicate copies of the SWI5 gene with potential subfunctionalization or neofunctionalization.

How is the SWI5 gene conserved across species?

The SWI5 gene demonstrates significant evolutionary conservation from yeasts to higher vertebrates. In budding yeast, the Sae3-Mei5 complex functions specifically during meiosis and interacts with Dmc1, while in fission yeast, the Swi5-Sfr1 complex plays a critical role in homologous recombination repair . The human SWI5-MEI5 complex (formed by C9orf119-C10orf78 proteins) interacts directly with RAD51 in vitro and is essential for homologous recombination repair . Given these conserved interactions across distant evolutionary lineages, the Atlantic salmon SWI5 homolog likely maintains similar critical functions in DNA repair mechanisms.

What is the role of SWI5 in DNA repair mechanisms?

SWI5 primarily functions in homologous recombination (HR) repair, a pathway crucial for repairing double-strand breaks in DNA. Studies in model organisms and humans indicate that SWI5 forms a complex with MEI5, which facilitates the loading and stabilization of recombinases like RAD51 onto single-stranded DNA during the homologous recombination process . In human cells, depletion of either SWI5 or MEI5 results in defects in homologous recombination repair and enhanced sensitivity to ionizing radiation , suggesting a conserved role in the DNA damage response that likely extends to Atlantic salmon.

How does the whole-genome duplication in salmon affect the SWI5 gene?

The salmonid-specific fourth vertebrate whole-genome duplication (Ss4R) likely created duplicate copies of many genes, including SWI5 . According to research on the Atlantic salmon genome, large genomic reorganizations coincided with transposon-mediated repeat expansions, which were crucial for the post-Ss4R rediploidization process . Analysis of duplicate retention patterns in the salmon genome revealed that neofunctionalization (acquiring new functions) was more common than subfunctionalization (partitioning of ancestral functions) . Interestingly, genes retained as duplicates after the teleost-specific whole-genome duplication were not more likely to be retained after the Ss4R, suggesting complex mechanisms governing duplicate retention .

What are the structural characteristics of the Salmo salar SWI5 protein?

While specific structural data for the Salmo salar SWI5 protein is limited in available research, comparative analysis with human SWI5 suggests it likely contains a C-terminal Swi5 domain that mediates interaction with MEI5. In humans, the SWI5-MEI5 complex forms through interaction between the C-terminal Swi5 domain of SWI5 and the middle coiled-coil region of MEI5 . This interaction is likely conserved in the salmon homolog, given the evolutionary conservation of this complex across species.

How can recombinant Salmo salar SWI5 be expressed and purified for functional studies?

For expression and purification of recombinant Salmo salar SWI5, researchers should consider the following methodological approach:

• Gene identification and cloning: Identify the SWI5 sequence from the Atlantic salmon genome database . Design primers to amplify the coding sequence, considering potential duplicate genes resulting from the Ss4R whole-genome duplication.

• Expression system selection: Consider eukaryotic expression systems for proper folding and post-translational modifications. Options include:

  • Bacterial systems (E. coli) with fusion tags (His, GST) for simpler purification

  • Insect cell systems (baculovirus) for better folding of eukaryotic proteins

  • Yeast expression systems for cost-effective eukaryotic expression

• Co-expression considerations: Based on human SWI5-MEI5 interaction studies , co-expression with salmon MEI5 may be necessary to obtain a stable, functional complex.

• Purification strategy:

  • Affinity chromatography using the fusion tag

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography to isolate the properly folded protein/complex

• Functional validation: Assess DNA binding activity and interaction with known partners using biochemical assays.

What experimental approaches are most effective for studying SWI5 interactions with other DNA repair proteins in salmon?

To investigate SWI5 interactions with other DNA repair proteins in salmon, several complementary approaches are recommended:

• In vitro interaction studies:

  • Pull-down assays using recombinant salmon SWI5 as bait

  • Co-immunoprecipitation with antibodies against SWI5 or potential partners

  • Surface plasmon resonance to measure binding affinities

• Cell-based interaction studies:

  • Develop salmon cell lines expressing tagged SWI5

  • Use proximity ligation assays to visualize protein interactions

  • Apply FRET/BRET techniques to study interactions in living cells

• Identification of the interactome:

  • Mass spectrometry analysis of proteins co-purifying with SWI5

  • Yeast two-hybrid screening to identify novel interacting partners

• Focus on established partners:

  • Based on human studies, prioritize investigation of interactions with MEI5 and RAD51

  • Examine whether duplicate copies of SWI5 resulting from whole-genome duplication have different interaction profiles

How does the function of SWI5 in Atlantic salmon compare to its orthologs in other teleost fish?

Comparative analysis of SWI5 function across teleost species should include:

• Ortholog identification: Use reciprocal BLAST searches and synteny analysis to identify true orthologs across teleost species.

• Sequence comparison: Analyze conservation of functional domains, particularly the C-terminal Swi5 domain implicated in MEI5 interaction .

• Expression pattern analysis: Compare tissue-specific expression profiles, considering that the Atlantic salmon genome shows evidence of regulatory neofunctionalization in many duplicated genes .

• Functional comparison: Develop assays to compare DNA repair efficiency between salmon SWI5 and orthologs from other teleost species.

• Evolutionary analysis: Examine whether SWI5 duplicates in salmon show differential evolutionary rates compared to single-copy genes in other teleosts.

What is the impact of environmental stressors on SWI5 expression in Atlantic salmon?

To investigate environmental stress effects on SWI5 expression:

• Cell culture model: Utilize Atlantic salmon primary cell cultures, similar to the muscle cell culture approach described for inflammation studies , to examine SWI5 expression under controlled stress conditions.

• Stress conditions to test:

  • Temperature fluctuations relevant to climate change scenarios

  • Hypoxia conditions mimicking poor water quality

  • Exposure to environmental pollutants

  • Inflammatory stimuli, such as recombinant IL-1β

• Expression analysis:

  • Quantitative RT-PCR to measure changes in SWI5 mRNA levels

  • Western blotting to assess protein expression

  • RNA-seq for genome-wide expression changes

• Duplicate gene consideration: Determine whether SWI5 duplicates (resulting from Ss4R) respond differently to stressors, which would suggest subfunctionalization in stress response pathways.

How can CRISPR-Cas9 be used to investigate SWI5 function in salmon cells?

CRISPR-Cas9 approaches for studying SWI5 function in salmon cells should include:

• sgRNA design considerations:

  • Target conserved regions in SWI5

  • Account for potential duplicate genes resulting from the Ss4R whole-genome duplication

  • Design multiple sgRNAs to increase editing efficiency

• Delivery methods:

  • Optimize transfection protocols for salmon cell lines

  • Consider electroporation for primary cells

• Functional analysis of edited cells:

  • Assess DNA repair capacity using comet assays

  • Measure sensitivity to DNA-damaging agents

  • Analyze RAD51 foci formation following DNA damage

• Advanced applications:

  • Generate knock-in cell lines expressing tagged SWI5 for localization studies

  • Create specific mutations to test functional domains

  • Investigate the consequences of disrupting only one SWI5 duplicate vs. both duplicates

What are the implications of SWI5 duplication in the salmon genome for understanding evolutionary mechanisms of genome duplication?

The duplication of SWI5 in the salmon genome provides valuable insights into post-whole-genome duplication evolutionary processes:

• Retention patterns: Analyze whether SWI5 follows the general pattern observed in the salmon genome, where genes retained after the teleost-specific whole-genome duplication were not more likely to be retained after the Ss4R .

• Evolutionary fate analysis:

  • Subfunctionalization: Test whether duplicate copies show tissue-specific expression

  • Neofunctionalization: Investigate whether duplicates have acquired novel functions

  • Dosage selection: Examine whether duplicates are retained due to stoichiometric constraints

• Comparative analysis with other DNA repair genes:

  • Determine if DNA repair pathways show consistent duplication patterns

  • Analyze whether functionally related proteins (e.g., MEI5) show similar duplication patterns

• Selective pressure analysis: Calculate Ka/Ks ratios to detect evolutionary constraints on different protein domains.

How do different isoforms of SWI5 in Salmo salar differ in their functional properties?

Characterization of SWI5 isoforms in Atlantic salmon should include:

• Isoform identification:

  • Distinguish between duplicates resulting from whole-genome duplication

  • Identify splice variants through transcriptome analysis

• Expression pattern analysis:

  • Determine tissue-specific expression patterns

  • Analyze developmental regulation

  • Assess responses to environmental stimuli

• Functional comparison:

  • Compare DNA binding properties

  • Assess interaction with MEI5 and other partners

  • Evaluate ability to complement SWI5 deficiency in cellular models

• Regulation analysis:

  • Investigate whether isoforms show differential responses to DNA damage

  • Examine potential cross-talk between the immune system and SWI5 regulation, as suggested by studies of inflammatory responses in salmon muscle cells

What experimental controls are essential when studying recombinant Salmo salar SWI5?

Proper experimental controls are critical for ensuring reliable results:

• Expression and purification controls:

  • Include tag-only control proteins to account for tag effects

  • Use known DNA repair proteins as positive controls for functional assays

  • Include species-specific controls when comparing across evolutionary lineages

• Functional assay controls:

  • Use human SWI5-MEI5 complex as a reference for comparative studies

  • Include wild-type proteins alongside mutant variants

  • Employ both positive controls (known functional proteins) and negative controls (non-functional variants)

• Specificity controls:

  • Validate antibody specificity using knockout or knockdown cells

  • Perform competition assays to confirm binding specificity

  • Include cross-reactivity tests when studying closely related duplicates

How should researchers approach the analysis of SWI5 expression data in the context of the duplicated salmon genome?

When analyzing expression data for SWI5 in the duplicated salmon genome:

• Duplicate-specific analysis:

  • Design primers/probes that can distinguish between duplicate copies

  • Use RNA-seq data with appropriate bioinformatic pipelines that account for highly similar duplicates

• Context interpretation:

  • Consider tissue-specific expression patterns, as salmon shows extensive regulatory neofunctionalization

  • Analyze co-expression networks to identify functional relationships

• Evolutionary context:

  • Compare expression patterns with single-copy orthologs in other species

  • Assess whether expression divergence follows patterns observed for other duplicated genes in salmon

What are the challenges in developing antibodies specific to Salmo salar SWI5?

Developing specific antibodies for salmon SWI5 presents several challenges:

• Duplicate gene considerations:

  • Identify epitopes that differentiate between duplicate copies

  • Validate specificity using recombinant proteins from each duplicate

• Cross-reactivity testing:

  • Test against other teleost SWI5 proteins to assess species specificity

  • Evaluate potential cross-reactivity with other DNA repair proteins

• Validation approaches:

  • Use CRISPR knockout cells as negative controls

  • Perform peptide competition assays

  • Validate antibody performance in multiple applications (Western blot, IP, immunofluorescence)

How might understanding SWI5 function contribute to conservation efforts for wild Atlantic salmon populations?

Research on SWI5 could contribute to conservation by:

• Biomarker development:

  • Establish whether SWI5 expression or function can serve as a biomarker for environmental stress

  • Determine if genetic variations in SWI5 correlate with population resilience

• Genetic diversity assessment:

  • Analyze SWI5 sequence variation across wild populations

  • Determine whether certain variants confer advantages under specific environmental conditions

• Environmental impact assessment:

  • Investigate whether pollutants affect SWI5 function and DNA repair capacity

  • Determine if climate change-related stressors impact SWI5-mediated genome stability

What opportunities exist for comparative studies between Salmo salar SWI5 and SWI5 homologs in other commercially important aquaculture species?

Comparative studies could provide valuable insights for aquaculture:

• Cross-species comparisons:

  • Compare SWI5 function between salmon and other aquaculture species like rainbow trout, sea bass, or tilapia

  • Determine whether species differences in genome stability correlate with SWI5 function

• Stress response comparison:

  • Analyze whether SWI5 response to environmental stressors differs between species

  • Identify species-specific adaptations in DNA repair pathways

• Genome duplication implications:

  • Compare species with different histories of genome duplication

  • Assess whether genome duplication provides advantages in DNA repair capacity