Recombinant Ashbya gossypii DNA replication complex GINS protein PSF3 (PSF3)

Basic Research Questions

• What is the GINS complex in Ashbya gossypii and what role does PSF3 play within it?

  The GINS complex (named from the Japanese Go, Ichi, Nii, and San; meaning five, one, two, and three) is a novel multiprotein complex essential for chromosomal DNA replication in eukaryotic cells. In Ashbya gossypii, as in other eukaryotes, GINS consists of four proteins: Sld5, Psf1 (partner of Sld five 1), Psf2, and Psf3 .

  PSF3 functions as a crucial component of this tetrameric GINS complex that associates with replication origins during S phase and then with neighboring DNA sequences as replication progresses. The complex is required for both the initiation of DNA replication and for normal replication fork progression .

  Research has demonstrated that the abundance and composition of GINS remain constant throughout the cell cycle, indicating its stable nature. The PSF3 protein specifically corresponds to the AGR399C gene in A. gossypii .

• Why is Ashbya gossypii used as a model organism for DNA replication studies?

  Ashbya gossypii offers several distinctive advantages as a model organism for studying DNA replication:

  • Genomic similarity: A. gossypii shares extensive synteny (>90%) with Saccharomyces cerevisiae, making it valuable for comparative genomics .

  • Unique morphology: Unlike other filamentous fungi related to budding yeast, A. gossypii grows as multinucleated hyphae while maintaining a genome similar to budding yeast .

  • Genetic tractability: High efficiency of homologous recombination allows for precise genetic manipulation .

  • Complete genome sequencing: The availability of its genome sequence facilitates systematic studies .

  • Evolutionary insights: A. gossypii provides a unique window into understanding how similar genetic material can support different growth morphologies (unicellular vs. filamentous) .

  These features make A. gossypii particularly useful for studying fundamental DNA replication processes, which can then inform our understanding of more complex eukaryotic systems .

• What is the evolutionary conservation of PSF3 across different fungal species?

  PSF3 is highly conserved across eukaryotic species, indicating its essential role in DNA replication. Comparative analysis shows:

  Species	Gene Name	Gene ID	Similarity to A. gossypii PSF3
  A. gossypii	AGOS_AGR399C	AGR399C	100% (reference)
  S. cerevisiae	YOL146W	PSF3	High conservation
  E. cymbalariae	-	-	High conservation within Eremothecium genus

  The conservation of PSF3 extends beyond fungi to higher eukaryotes, indicating its fundamental role in DNA replication machinery. Within the Saccharomycetaceae family, to which A. gossypii belongs, the GINS complex proteins show significant functional conservation despite varying degrees of sequence homology .

  Recent genome sequencing of multiple Ashbya species, including A. aceri, revealed that genes involved in DNA replication, including PSF3, are among the most highly conserved between species, suggesting their essential function has been maintained throughout evolution .

Advanced Research Questions

• How does the GINS complex interact with other DNA replication proteins in Ashbya gossypii?

  The GINS complex in A. gossypii functions as part of a larger macromolecular assembly engaged in DNA replication. Key interactions include:

  • Dpb11 and Sld3-Cdc45 interactions: GINS interacts with both Dpb11 and the Sld3-Cdc45 complex in a mutually dependent manner . Without GINS, neither Dpb11 nor Cdc45 associates properly with chromatin DNA.

  • MCM complex interactions: BiFC (Bimolecular Fluorescence Complementation) studies in the related fungus Schizosaccharomyces pombe have visualized direct interactions between GINS and MCM proteins in nuclei of growing cells and on chromatin during S-phase .

  • Component interdependence: Experimental evidence shows that without Dpb11 or Sld3, GINS fails to associate with replication origins, indicating a coordinated assembly mechanism .

  Two-hybrid assays have specifically demonstrated that the Psf1 component of GINS interacts with Dpb2, Dpb11, Sld3, and Sld5, providing a molecular basis for how GINS mediates between the Sld3–Cdc45 and Dpb11–Pol ɛ complexes .

  The current model suggests GINS acts as a crucial bridge that facilitates proper association between origins and DNA polymerases during initiation of DNA replication.

• What methodologies can be used to study the function of recombinant A. gossypii PSF3 in vitro?

  Several methodologies are particularly effective for studying recombinant A. gossypii PSF3:

  Protein Expression and Purification:

  • Expression systems: E. coli, yeast (S. cerevisiae), baculovirus, or mammalian cell expression systems

  • Purification to ≥85% purity using affinity chromatography followed by ion exchange and size exclusion chromatography

  • Tagging strategies: N-terminal or C-terminal His-tags facilitate purification while minimizing interference with protein function

  Functional Assays:

  • DNA binding assays: Electrophoretic mobility shift assays (EMSA) to assess PSF3's ability to bind DNA directly or as part of the GINS complex

  • Helicase loading assays: Measuring PSF3/GINS contribution to MCM helicase loading on DNA templates

  • Reconstitution experiments: Assembly of the complete GINS complex from recombinant components to study tetramer formation and stability

  Structural Studies:

  • X-ray crystallography of recombinant PSF3 alone or as part of the GINS complex

  • Cryo-EM to visualize larger assemblies containing PSF3/GINS

  Interaction Studies:

  • Pull-down assays using tagged PSF3 to identify interaction partners

  • Surface plasmon resonance (SPR) to measure binding kinetics with known partners

• How can mutations in the PSF3 gene affect DNA replication and cell viability in A. gossypii?

  Mutations in PSF3 can have profound effects on DNA replication and cell viability in A. gossypii, similar to observations in related yeasts:

  Temperature-sensitive mutations:
  Studies with thermosensitive mutants of GINS components (including PSF3) have demonstrated that under restrictive conditions, these mutations lead to defects in DNA replication, causing :

  • Failure to initiate DNA replication at origins

  • Impaired progression of replication forks

  • Cell cycle arrest

  • Ultimately, loss of cell viability

  Structural consequences:
  The stability of the entire GINS complex is compromised when individual components are mutated. For example, when Sld5 (another GINS component) was mutated, the amount of GINS complex recovered was reduced to less than 1/5 of that from wild-type cells, and uncomplexed Psf1 was degraded . Similar destabilization effects would be expected for PSF3 mutations.

  Suppression analysis:
  Interestingly, some temperature-sensitive mutations in GINS components can be suppressed by overexpression of other GINS components. This suggests that stabilizing the complex formation can overcome certain defects, providing insight into the functional relationships between components .

• What techniques can be used to visualize PSF3-protein interactions in vivo in A. gossypii?

  Several advanced techniques can be employed to visualize PSF3-protein interactions in A. gossypii:

  Bimolecular Fluorescence Complementation (BiFC):

  • This technique has been successfully applied in the related fungus S. pombe to visualize interactions between GINS and MCM subunits

  • For A. gossypii, BiFC can be implemented by tagging PSF3 with one half of a split fluorescent protein (e.g., YFP) and a potential interaction partner with the complementary half

  • When the proteins interact, the fluorescent protein fragments come together to produce a detectable signal

  • This allows visualization of both the occurrence and subcellular localization of interactions

  Fluorescence Resonance Energy Transfer (FRET):

  • By tagging PSF3 and its interaction partners with appropriate donor and acceptor fluorophores (e.g., CFP and YFP)

  • Requires more sophisticated imaging equipment than BiFC but provides dynamic interaction information

  Chromatin Immunoprecipitation (ChIP):

  • To analyze PSF3 association with chromatin during DNA replication

  • Can be combined with next-generation sequencing (ChIP-seq) to map genome-wide binding sites

  Proximity Ligation Assay (PLA):

  • Detects protein interactions with high sensitivity and specificity

  • Particularly useful for detecting endogenous protein interactions without overexpression

  These techniques can be applied to study both constitutive and cell cycle-regulated interactions of PSF3 with other replication proteins in the natural cellular environment of A. gossypii.

Methodological Questions

• What are the optimal conditions for expressing and purifying recombinant A. gossypii PSF3?

  The optimal conditions for expressing and purifying recombinant A. gossypii PSF3 based on established protocols include:

  Expression Systems:

  Expression System	Advantages	Yield	Purity
  E. coli	Rapid growth, high yield	High	≥85%
  Yeast	Eukaryotic PTMs	Medium	≥85%
  Baculovirus	Complex proteins	Medium	≥85%
  Mammalian cells	Native folding	Low	≥85%

  Optimal Expression Protocol:

  1. Clone the AGR399C gene into an appropriate expression vector with a His-tag (N- or C-terminal)

  2. For E. coli expression: Transform into BL21(DE3) strain

  3. Grow cultures at 37°C until OD600 reaches 0.6-0.8

  4. Induce protein expression with 0.5mM IPTG

  5. Shift temperature to 18°C for overnight expression to enhance solubility

  Purification Strategy:

  1. Lyse cells in buffer containing 50mM Tris-HCl pH 8.0, 300mM NaCl, 10% glycerol, 1mM DTT, protease inhibitors

  2. Initial purification via Ni-NTA affinity chromatography

  3. Further purification using ion exchange chromatography

  4. Final polishing step with size exclusion chromatography

  5. The purified protein should reach ≥85% purity as determined by SDS-PAGE

  Co-expression Strategy:
  For functional studies, co-expression of all four GINS components (Sld5, Psf1, Psf2, and PSF3) may yield more biologically relevant material, as the GINS complex functions as a tetramer.

• How can I design an effective experimental system to study PSF3 function in chromosomal DNA replication?

  An effective experimental system to study PSF3 function should address multiple aspects of replication complex biology:

  Genetic Approaches:

  1. Temperature-sensitive mutants: Create conditional PSF3 mutants in A. gossypii using site-directed mutagenesis. This has been successfully used for GINS components, revealing their essential roles in DNA replication .

  2. Depletion systems: Implement an auxin-inducible degron system for rapid depletion of PSF3 to observe immediate effects on replication.

  3. Domain dissection: Generate truncation mutants to map functional domains of PSF3 involved in complex formation and chromatin binding.

  Biochemical Approaches:

  1. Reconstitution assays: Assemble the GINS complex and larger replication assemblies in vitro using purified components to study PSF3's contribution.

  2. Pull-down assays: Use recombinant tagged PSF3 to identify interaction partners through mass spectrometry.

  3. Binding assays: Characterize interactions of PSF3 with other replication proteins and DNA using techniques like SPR or MST.

  Cell Biology Approaches:

  1. Live cell imaging: Use fluorescently-tagged PSF3 to track its localization during the cell cycle in A. gossypii.

  2. BiFC or FRET: As described in question 7, these techniques can visualize protein interactions in vivo .

  3. Chromatin association: Use ChIP-seq to map PSF3 binding sites across the genome during replication.

  Systems-level Approaches:

  1. Genomics: RNA-seq analysis following PSF3 depletion to identify affected pathways.

  2. Proteomics: Study changes in the replication complex composition when PSF3 is mutated.

  3. Synthetic genetic interactions: Screen for genetic interactions with PSF3 to place it in functional networks.

• What are the differences in PSF3 function between A. gossypii and closely related yeasts like Saccharomyces cerevisiae?

  While PSF3 serves the fundamental role of DNA replication in both organisms, there are notable differences between A. gossypii and S. cerevisiae:

  Genomic Context:

  • Despite the high synteny (>90%) between A. gossypii and S. cerevisiae genomes , the genomic context of PSF3 differs between these organisms.

  • In A. gossypii, PSF3 is encoded by the AGOS_AGR399C gene , while in S. cerevisiae, it is encoded by YOL146W.

  Growth Pattern Implications:

  • A key difference is that A. gossypii grows as multinucleated hyphae, unlike the unicellular S. cerevisiae .

  • This means that DNA replication in A. gossypii must be coordinated across multiple nuclei within the same cytoplasm, potentially requiring adaptations in the GINS complex function.

  Replication Dynamics:

  • The filamentous growth of A. gossypii likely requires specialized regulation of DNA replication origins and timing.

  • Studies comparing origin activation and replication fork progression between these species could reveal adaptations of PSF3/GINS function to different growth modes.

  Protein Interactions:

  • While the core interactions of PSF3 within the GINS complex are conserved, the specific protein-protein interactions may vary between species.

  • Two-hybrid assays have identified interactions between GINS components and other replication proteins that may have species-specific characteristics .

  Understanding these differences provides valuable insights into how conserved replication machinery has been adapted to support different growth morphologies during evolution.

• How can recombinant A. gossypii PSF3 be used as a tool to study DNA replication mechanisms?

  Recombinant A. gossypii PSF3 serves as a valuable tool for investigating DNA replication through multiple experimental approaches:

  Structural Studies:

  • Purified recombinant PSF3 can be used for crystallography or cryo-EM studies to determine its structure alone or within the GINS complex

  • Structural information provides insights into functional domains and interaction interfaces

  Biochemical Reconstitution:

  • Recombinant PSF3 enables in vitro reconstitution of the GINS complex

  • Reconstituted complexes can be used to study:

    • Assembly and stability of replication complexes

    • DNA binding properties

    • Helicase loading and activation mechanisms

  Interaction Studies:

  • Pull-down assays using recombinant PSF3 as bait to identify novel interaction partners

  • Competition assays to map binding interfaces between PSF3 and known partners

  • In vitro validation of interactions identified through genetic or proteomic approaches

  Antibody Generation:

  • Recombinant PSF3 can be used to generate specific antibodies for:

    • Immunoprecipitation studies

    • ChIP experiments to map genomic binding sites

    • Western blotting to study expression levels and modifications

  Template for Mutation Analysis:

  • Site-directed mutagenesis of recombinant PSF3 allows systematic analysis of:

    • Critical residues for complex formation

    • DNA binding interfaces

    • Post-translational modification sites

  By providing a pure, consistent source of PSF3 protein, researchers can conduct detailed mechanistic studies that would be difficult or impossible using only genetic approaches.

• What challenges are associated with expressing functional recombinant PSF3, and how can they be overcome?

  Producing functional recombinant PSF3 presents several challenges that researchers should address:

  Challenge 1: Protein Solubility

  • PSF3 may form inclusion bodies when overexpressed, particularly in bacterial systems

  • Solutions:

    • Lower induction temperature (18-20°C) during expression

    • Use solubility-enhancing fusion tags (MBP, SUMO, or thioredoxin)

    • Optimize buffer conditions with solubility enhancers like glycerol (10-15%) or low concentrations of non-ionic detergents

  Challenge 2: Complex Dependency

  • PSF3 naturally functions as part of the GINS tetramer, and isolated PSF3 may be unstable or non-functional

  • Solutions:

    • Co-express all four GINS components (Sld5, Psf1, Psf2, and PSF3)

    • Use a polycistronic expression vector or dual plasmid systems

    • Sequential purification strategy incorporating multiple affinity tags

  Challenge 3: Post-translational Modifications

  • Bacterial expression systems lack eukaryotic post-translational modifications

  • Solutions:

    • Express in eukaryotic systems (yeast, insect, or mammalian cells)

    • Identify and characterize relevant modifications in native PSF3

    • Consider chemical or enzymatic post-expression modification if critical

  Challenge 4: Structural Integrity Verification

  • Ensuring the recombinant protein adopts the correct folding

  • Solutions:

    • Circular dichroism spectroscopy to assess secondary structure

    • Limited proteolysis to evaluate domain organization

    • Functional assays to confirm activity (DNA binding, complex formation)

  Challenge 5: Stability During Storage

  • Purified PSF3 may aggregate or lose activity during storage

  • Solutions:

    • Optimize buffer conditions (pH, salt, reducing agents)

    • Add stabilizers like glycerol (10-20%)

    • Flash-freeze in liquid nitrogen and store at -80°C in small aliquots

    • Test activity after storage to ensure functionality is maintained