Recombinant Candida glabrata Mitochondrial morphogenesis protein SLD7 (SLD7)

What is the primary function of SLD7 in Candida glabrata?

SLD7 appears to have a significant role in chromosomal DNA replication in yeast species. Although not essential for cell viability, research indicates it plays an important function in ensuring efficient DNA replication . While the search results don't explicitly confirm its role in mitochondrial morphogenesis in C. glabrata, the protein forms a tight complex with Sld3 throughout the cell cycle, which affects replication timing and efficiency . Methodologically, this was determined through genetic interaction studies and flow cytometry analysis measuring DNA content in wild-type versus sld7Δ cells.

How does SLD7 interact with other proteins in the replication pathway?

SLD7 primarily interacts by forming a tight complex with Sld3, which in turn associates with replication origins in a complex with Cdc45 . This interaction was confirmed through two-step immunoprecipitation techniques using Sld7 protein tandemly tagged with 3Flag-HA. Mass spectrometry analysis of the coprecipitated proteins identified Sld3 as an 80-kDa protein that specifically binds to Sld7 . Additionally, SLD7 binds to the non-essential N-terminal portion of Sld3 and affects its affinity for Cdc45, which is a component of the replication fork .

What happens in cells when SLD7 is deleted?

Deletion of SLD7 (sld7Δ) has several measurable consequences:

• Reduced cellular levels of Sld3

• Delayed dissociation of GINS (a replication fork component) from origins

• Significantly slowed S-phase progression

• Increased sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS)

• Extended time to complete DNA replication (approximately twice as long as wild-type cells)

Flow cytometry analysis showed that while cell-cycle events at the G1/S boundary appear normal in sld7Δ cells, DNA replication takes about twice as long to complete compared to wild-type cells .

What are the most effective methods for expressing recombinant SLD7 protein?

Based on available research approaches, recombinant SLD7 expression would likely follow protocols similar to those used for other yeast proteins. An effective approach would include:

• Gene cloning into expression vectors with appropriate tags (e.g., 3Flag-HA as used in the research)

• Expression in either E. coli or yeast expression systems

• Purification using affinity chromatography based on the introduced tags

The research demonstrated successful expression and tagging of Sld7 with 3Flag-HA tags for immunoprecipitation studies , suggesting this approach is viable for recombinant expression.

What assays can be used to study SLD7's function in DNA replication?

Several experimental approaches can be employed:

• Flow cytometry analysis: To measure DNA content and replication timing in wild-type versus sld7Δ cells

• Genetic interaction studies: Testing synthetic lethality with mutations in other replication genes (e.g., DPB11, SLD genes, Pol ε, GINS)

• Protein-protein interaction assays:

  • Two-step immunoprecipitation using tagged proteins

  • Two-hybrid assays to map interaction domains

• Drug sensitivity assays: Testing growth on plates containing hydroxyurea or methyl methanesulfonate

• Cell synchronization experiments: Using α-factor arrest and release to study S-phase progression

How can researchers confirm SLD7-Sld3 interactions in experimental settings?

The research highlights several complementary approaches:

• Two-step immunoprecipitation: Using tandem affinity tags (3Flag-HA) on Sld7 to isolate protein complexes, followed by mass spectrometry to identify binding partners

• Two-hybrid assays: Used effectively to map the Sld7-binding region of Sld3

• Co-immunoprecipitation: Directly testing protein-protein interactions in cell extracts

• Protein stability assays: Measuring Sld3 levels in the presence or absence of Sld7 to confirm stabilization effects

How does SLD7 coordinate with the cell cycle machinery to regulate DNA replication timing?

While the exact mechanisms remain to be fully elucidated, research indicates that Sld7 forms a complex with Sld3 throughout the cell cycle . This complex associates with replication origins along with Cdc45. Importantly, Sld3 binds to Dpb11 when phosphorylated by cyclin-dependent kinase, suggesting cell-cycle regulation . The complex dissociates from origins once DNA replication starts, but interestingly, Sld7 does not move with the replication fork . This suggests a role in initiation rather than elongation of DNA replication.

Advanced research methodologies to explore this question would include:

• Chromatin immunoprecipitation (ChIP) to track protein-DNA associations throughout the cell cycle

• Phosphoproteomics to identify regulatory phosphorylation events

• Single-molecule imaging to visualize protein dynamics during replication

What is the molecular mechanism by which SLD7 affects Sld3's affinity for Cdc45?

This represents a key mechanistic question. The research indicates that Sld7 binds to the non-essential N-terminal portion of Sld3 and reduces its affinity for Cdc45 . To fully understand this mechanism, researchers should consider:

• Structural biology approaches:

  • X-ray crystallography or cryo-EM of the Sld7-Sld3 complex

  • NMR studies of the interaction domains

• Biochemical approaches:

  • In vitro binding assays with purified components

  • Mutational analysis of binding interfaces

  • Isothermal titration calorimetry to measure binding affinities

• Computational approaches:

  • Molecular dynamics simulations

  • Protein-protein docking

How might the function of SLD7 differ between Candida glabrata and other fungal species?

This question addresses important evolutionary and comparative aspects. While the search results don't directly compare C. glabrata SLD7 with homologs in other species, the data on Sld7 in yeast provides a framework for comparison . Researchers investigating this question should:

• Perform phylogenetic analyses of SLD7 homologs across fungal species

• Conduct complementation studies to determine functional conservation

• Compare protein-protein interaction networks between species

• Examine differential expression patterns and regulation

• Assess phenotypic consequences of deletion in different species

Could SLD7 be a potential target for antifungal development against Candida glabrata?

While SLD7 is not essential for viability, its deletion significantly impairs cell growth and DNA replication efficiency . This suggests potential as a target for developing drugs that would not kill C. glabrata outright but might significantly impair its growth and virulence.

A methodological approach to investigate this would include:

• High-throughput screening for small molecule inhibitors of Sld7-Sld3 interaction

• Structure-based drug design targeting the Sld7 protein

• Testing candidate compounds in combination with existing antifungals for synergistic effects

• In vivo infection models to assess efficacy of targeting SLD7

What is the relationship between SLD7's role in DNA replication and potential functions in mitochondrial morphogenesis?

This question addresses a potential dual role for SLD7. The search results focus on its role in nuclear DNA replication , but the query mentions mitochondrial morphogenesis. To investigate this potential dual function:

• Conduct subcellular localization studies using fluorescently tagged SLD7

• Examine mitochondrial morphology and function in sld7Δ cells

• Perform mitochondrial DNA (mtDNA) replication assays in the presence and absence of SLD7

• Use BioID or proximity labeling approaches to identify mitochondrial interaction partners

• Create separation-of-function mutants that affect only nuclear or only mitochondrial functions

How does SLD7 contribute to genome stability and stress response in Candida glabrata?

The research shows that sld7Δ cells have reduced viability when exposed to DNA damaging agents like hydroxyurea and methyl methanesulfonate . This suggests a broader role in genome stability. Researchers can investigate this through:

• Measuring mutation rates and chromosomal rearrangements in sld7Δ strains

• Examining DNA damage checkpoint activation in response to various stressors

• Analyzing genetic interactions with DNA repair pathways

• Conducting genome-wide sequencing of evolved sld7Δ strains to identify compensatory mutations

• Testing sensitivity to a broader range of genotoxic agents and environmental stressors

What are common pitfalls when working with recombinant SLD7 protein expression and purification?

Based on the experimental approaches described in the research, potential challenges include:

• Protein solubility: SLD7 may form insoluble aggregates when overexpressed

  • Solution: Optimize expression conditions (temperature, induction time)

  • Use solubility tags like MBP or SUMO

  • Consider native purification from yeast rather than bacterial expression

• Maintaining protein-protein interactions: The functional unit appears to be the SLD7-SLD3 complex

  • Solution: Co-express SLD7 with SLD3

  • Purify the intact complex rather than individual proteins

• Protein stability: Ensuring the recombinant protein maintains its native conformation

  • Solution: Include appropriate buffers and stabilizing agents

  • Validate function through in vitro activity assays

How can researchers overcome challenges in studying SLD7 function in vivo?

The research indicates several methodological approaches:

• Genetic redundancy: While SLD7 is non-essential, its deletion causes significant phenotypes

  • Solution: Create conditional alleles or degron-tagged versions for acute depletion

  • Identify and simultaneously target redundant pathways

• Phenotypic analysis: The effects of SLD7 deletion may be subtle or condition-dependent

  • Solution: Combine with mutations in interacting pathways to enhance phenotypes

  • Use sensitive assays like competitive growth or single-cell analysis

  • Test under various stress conditions

• Distinguishing direct from indirect effects:

  • Solution: Use rapid protein depletion systems rather than gene deletion

  • Complement with in vitro reconstitution of key activities