Recombinant Neurospora crassa DNA replication regulator sld-2 (sld-2), partial

What is the function of sld-2 in Neurospora crassa?

sld-2 functions as an essential DNA replication regulator in Neurospora crassa, playing a critical role in the initiation of DNA replication. Similar to its yeast homolog, N. crassa sld-2 is involved in the formation of replication complexes necessary for the accurate and timely replication of the fungal genome. The protein is well conserved in yeasts and fungi, with the sld-2 region representing one of the most conserved areas among sld-2 homologs .

Research indicates that sld-2, like its counterparts in other organisms, participates in the assembly of the replicative helicase. Studies in related systems have shown that when sld-2 is depleted or mutated, cells exhibit severe defects in DNA replication, illustrating its essential nature .

How is the structure-function relationship understood in sld-2?

The N. crassa sld-2 protein contains conserved domains that are crucial for its function. Based on homology studies with other fungal species:

• The protein contains regions responsible for DNA binding

• It has domains that mediate protein-protein interactions with other replication factors

• It contains multiple CDK phosphorylation sites that regulate its activity

Research in budding yeast has shown that Sld2 has two critical homology regions which may be conserved in N. crassa: one near the N-terminus (region I) and another at the C-terminus (region II). Interestingly, some newly identified Sld2 homologs appear to lack homology region II, while others lack region I, suggesting functional diversity across species .

What experimental techniques are commonly used to study sld-2 in Neurospora crassa?

Several methodological approaches have proven effective for studying sld-2:

• Recombinant protein expression: E. coli, yeast, baculovirus, or mammalian cell systems can be used for expressing recombinant N. crassa sld-2 with a purity greater than 85% as determined by SDS-PAGE .

• CRISPR/Cas9 gene editing: A user-friendly CRISPR/Cas9 system has been developed for N. crassa mutagenesis, allowing for efficient targeted modifications of the sld-2 gene. This system incorporates the cas9 sequence into the fungal genome and introduces guide RNA via electroporation, eliminating the need for constructing multiple vectors .

• Phosphorylation assays: In vitro phosphorylation assays using recombinant CDK can determine the phosphorylation status of sld-2 and its effect on interactions with other proteins .

• Two-hybrid assays: These can be used to study protein-protein interactions between sld-2 and other replication factors .

• DNA replication assays: Pulse-labeling with nucleoside analogs like 5-iodo-2′-deoxyuridine (IdU) followed by immunofluorescence visualization can assess replication defects after sld-2 depletion or mutation .

How does phosphorylation regulate sld-2 function in DNA replication?

Phosphorylation plays a critical regulatory role in sld-2 function, similar to what has been observed in homologous proteins in other organisms. Studies in related systems have revealed:

• Multiple phosphorylation sites: Similar to Sld2 in budding yeast, N. crassa sld-2 likely contains multiple CDK phosphorylation sites that regulate its activity. In yeast Sld2, 11 clustered CDK-phosphorylation motifs are present, six of which match canonical sequences .

• Threshold mechanism: Research suggests that multisite phosphorylation sets a threshold for activation. In yeast, phosphorylation of multiple sites is required for the critical phosphorylation of a specific residue (Thr84 in S. cerevisiae Sld2) .

• Conformational changes: Phosphorylation likely induces conformational changes in sld-2 that enable its interaction with other replication factors. This appears to be a conserved mechanism across species .

• Cell cycle control: CDK-dependent phosphorylation ensures that DNA replication initiation occurs only at the appropriate time during the cell cycle .

Research in C. elegans has shown that SLD-2 has eight CDK consensus sites, and mutation of these sites affects protein mobility and function, suggesting a similar regulatory mechanism may exist in N. crassa .

What is known about sld-2 interactions with other replication factors in fungi?

Based on homologous systems, N. crassa sld-2 likely interacts with several other replication factors:

Interaction with Dpb11 homolog: In yeast, phosphorylated Sld2 interacts with Dpb11 through a specific 20-amino-acid stretch that is one of the most conserved regions among Sld2 homologs. This interaction is essential for DNA replication and requires prior phosphorylation by CDK .

Interaction with MCM complex: Studies in budding yeast identified a mutant of Sld2 (Sld2-m1,4) that is specifically defective in Mcm2-7 binding. This mutation caused severe inhibition of DNA replication, suggesting that Sld2 binding to Mcm2-7 is essential to prevent inappropriate formation of the CMG helicase complex .

GINS complex interaction: Sld2 is involved in the assembly of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex. Research has shown that mutations in Sld2 that affect its DNA-binding capability result in defective GINS-Mcm2-7 interaction, indicating that Sld2's association with DNA is required for CMG assembly .

The table below summarizes key protein interactions observed in model fungal systems:

How does sld-2 contribute to origin efficiency versus timing in replication?

Research on the role of limiting DNA replication initiation factors like Sld2 and Sld3 has provided insights into how these factors affect replication origin activation:

• Impact on origin efficiency: Studies show that reduced abundance of Sld2 results in viability defects and genomic instability. When Sld2 is depleted, origin firing is depressed across the genome, indicating that Sld2 abundance primarily impacts origin efficiency rather than the timing of replication initiation .

• Chromosome stability: Depletion of Sld2 leads to significant instability of specific chromosomes. For example, Chromosome XII in yeast becomes particularly unstable due to the failure to complete rDNA replication. After Sld2 depletion, this chromosome may become trapped and fragmented, never completing replication .

• Timing preservation: Interestingly, even when initiation at specific origins (like rDNA origins) is impaired due to Sld2 depletion, these origins can still fire at their normal time in S phase. This suggests that Sld2 controls the efficiency (how many origins fire) rather than the timing of when specific origins fire .

These findings have implications for the ongoing debate surrounding the "stochastic model for origin firing" in the DNA replication field, suggesting that the abundance of factors like Sld2 creates a probabilistic landscape for origin activation rather than a strictly deterministic timing program .

How can CRISPR/Cas9 be used for functional studies of sld-2 in Neurospora crassa?

CRISPR/Cas9 offers a powerful approach for studying sld-2 function in N. crassa:

• System design: A user-friendly CRISPR/Cas9 system has been developed for N. crassa, where the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. This eliminates the need for constructing multiple vectors .

• Targeting strategy: For editing sld-2, researchers can design guide RNAs (gRNAs) targeting specific regions of the sld-2 sequence. The design should consider:

  • Target specificity

  • Minimal off-target effects

  • Proximity to functional domains

• Co-targeting approach: When studying non-selectable genes like sld-2, researchers can combine gRNAs targeting both sld-2 and a selectable marker gene like csr-1 (cyclosporin-resistant-1). This approach has been shown to increase the probability of finding mutants carrying the desired non-selectable mutation by up to tenfold .

• Mutation verification: After transformation, researchers should verify mutations through PCR amplification of the target region followed by sequencing. Common mutation types include small insertions (1-2 bp), small deletions (1-2 bp), and larger indels .

• Efficiency considerations: The editing efficiency of CRISPR/Cas9 in N. crassa ranges from 7.35% to 11.89% depending on the gRNA used, comparable to homologous recombination efficiency in a wild-type background .

What expression systems are optimal for producing recombinant Neurospora crassa sld-2?

Several expression systems can be used for producing recombinant N. crassa sld-2, each with specific advantages:

• E. coli expression system:

  • Advantages: Rapid growth, high yield, cost-effective

  • Considerations: May lack post-translational modifications, potential folding issues

  • Typical purification tag: His-tag or GST-tag

  • Expected purity: ≥85% as determined by SDS-PAGE

• Yeast expression system:

  • Advantages: Eukaryotic post-translational modifications, natural folding environment

  • Considerations: Lower yield than bacterial systems, longer production time

  • Suitable for studying phosphorylation: Can reproduce CDK-dependent phosphorylation

  • Applications: Particularly useful for protein-protein interaction studies

• Baculovirus expression:

  • Advantages: High yield of complex eukaryotic proteins, proper folding

  • Considerations: More technically demanding, higher cost

  • Applications: Ideal for structural studies requiring large amounts of properly folded protein

• Mammalian cell expression:

  • Advantages: Most sophisticated post-translational modifications

  • Considerations: Highest cost, lower yield, complex setup

  • Applications: Studies requiring precise mimicking of in vivo modifications

For functional studies of N. crassa sld-2, the yeast expression system might be particularly valuable as it allows observation of CDK-dependent phosphorylation similar to what occurs in vivo, as demonstrated by studies of homologous proteins .

What techniques can be used to study sld-2 phosphorylation and its effect on protein interactions?

Several methodological approaches can effectively assess sld-2 phosphorylation and its functional consequences:

• In vitro phosphorylation assays:

  • Use recombinant CDK (e.g., Cdc28–Clb5 in yeast) to phosphorylate purified sld-2

  • Monitor mobility shift in SDS-PAGE (phosphorylated proteins often migrate slower)

  • Quantify phosphorylation using radioactive ATP or phospho-specific antibodies

• Phosphorylation site mapping:

  • Create alanine substitutions at CDK-phosphorylation motifs (Ser/Thr–Pro)

  • Use phospho-specific antibodies to detect phosphorylation at specific sites

  • Employ mass spectrometry to identify all phosphorylated residues

• Protein-protein binding assays:

  • GST pull-down assays using phosphorylated and non-phosphorylated sld-2

  • Co-immunoprecipitation to detect interactions in vivo

  • Surface plasmon resonance to measure binding kinetics and affinity

• Functional assays:

  • Create phosphomimetic mutants (e.g., Ser/Thr→Asp substitutions)

  • Test complementation of sld-2 mutations by wild-type and mutant proteins

  • Assess effects on DNA replication through techniques like IdU labeling

• Two-hybrid assays:

  • Test interactions between sld-2 and potential binding partners

  • Compare interactions with wild-type, phospho-deficient, and phosphomimetic mutants

  • Map interaction domains through truncation analysis

Studies in yeast have shown that phosphorylation of multiple sites in Sld2 regulates a single specific phosphorylation event (at Thr84), which is essential for interaction with Dpb11. Similar regulatory mechanisms might exist in N. crassa sld-2 .

How can researchers study the role of sld-2 in replication origin efficiency?

To investigate how sld-2 affects replication origin efficiency in N. crassa, researchers can employ the following methodological approaches:

• Controlled protein depletion:

  • Use degron-tagged sld-2 for rapid protein degradation

  • Employ auxin-inducible degradation systems for temporal control

  • Monitor depletion through western blotting or fluorescent tagging

• Genome-wide replication analysis:

  • Pulse-label replicating DNA with nucleoside analogs (BrdU, EdU, IdU)

  • Perform DNA combing or DNA fiber analysis to visualize individual replication events

  • Use next-generation sequencing to map replication progression genome-wide

• Origin efficiency quantification:

  • Compare origin firing in wild-type versus sld-2-depleted cells

  • Measure the percentage of cells in which a specific origin fires

  • Calculate origin efficiency across multiple cell cycles

• Chromosome stability assessment:

  • Perform pulsed-field gel electrophoresis to monitor chromosome integrity

  • Track specific chromosomes using FISH (Fluorescence In Situ Hybridization)

  • Quantify chromosome loss rates using genetic markers

• Replication timing analysis:

  • Synchronize cells and collect samples at defined time points during S phase

  • Sequence newly replicated DNA to determine replication timing domains

  • Compare timing profiles between wild-type and sld-2-mutant cells

Research in yeast has shown that reduced abundance of Sld2 affects origin efficiency rather than timing, with specific chromosomal regions (like rDNA) being particularly sensitive to Sld2 depletion .

What approaches can be used to investigate sld-2's role in preventing premature helicase assembly?

Based on studies of homologous proteins, the following methodological approaches can be used to study sld-2's role in preventing premature helicase assembly:

• Interaction-defective mutants:

  • Generate sld-2 mutants specifically defective in Mcm2-7 binding (similar to the Sld2-m1,4 mutant in yeast)

  • Create DNA-binding defective mutants (like sld2-DNA)

  • Express these mutants and assess their effects on replication and cell viability

• Helicase assembly monitoring:

  • Use co-immunoprecipitation to detect formation of the CMG (Cdc45-Mcm2-7-GINS) complex

  • Perform chromatin immunoprecipitation to assess helicase components at origins

  • Compare assembly timing between wild-type and mutant cells

• Cell cycle analysis:

  • Synchronize cells at different cell cycle stages

  • Monitor helicase assembly specifically in G1 versus S phase

  • Determine if sld-2 mutations cause premature CMG formation in G1

• Genetic interaction studies:

  • Combine sld-2 mutations with mutations in other replication factors

  • Look for synthetic lethality or suppression

  • Identify genetic pathways that work in parallel with sld-2

Research in yeast has shown that an Sld2-Mcm2-7 binding mutant exhibits premature helicase assembly in G1, while an Sld2-DNA binding mutant displays no helicase assembly at all. This suggests that Sld2-Mcm interaction is critical to prevent premature assembly of the replicative helicase, while Sld2-DNA interaction is required for proper helicase assembly .

How conserved is sld-2 across fungal species and what does this tell us about its function?

The conservation pattern of sld-2 across fungal species provides important insights into its evolutionary significance and functional domains:

• Conservation across fungi: sld-2 is well conserved across various fungal species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe (where it's called drc1), Neurospora crassa, Aspergillus species, and many others .

• Homology regions: Alignment studies have identified two key homology regions in Sld2 proteins:

  • Region I: Located near the N-terminus, this region is one of the most conserved areas among Sld2 homologs

  • Region II: Located at the C-terminus

  • Interestingly, some newly identified Sld2 homologs appear to lack homology region II (e.g., in Trypanosoma cruzi and Cryptococcus neoformans), while others lack region I (e.g., in Arabidopsis thaliana)

• Functional conservation: The conservation of key regions suggests functional importance. For example:

  • A mutation in the fission yeast Drc1 (P112S), corresponding to the sld2-6 mutation in budding yeast, confers defective cell growth

  • The 20-amino-acid stretch involved in binding to Dpb11 is highly conserved across species, indicating the importance of this interaction

• Structural requirements: When the amino-acid sequence between Thr84 and Ser100 in budding yeast Sld2 was replaced with the corresponding sequence from fission yeast Drc1, the hybrid Sld2 interacted with the fission yeast Dpb11 homolog (Cut5) instead of Dpb11, demonstrating the specificity of partner binding is encoded in this region .

The evolutionary conservation of sld-2 across fungal species highlights its fundamental role in DNA replication, while the variation in specific regions suggests adaptation to species-specific replication mechanisms.

How does N. crassa sld-2 compare to homologs in other model organisms?

Comparative analysis reveals both similarities and differences between N. crassa sld-2 and its homologs in other model organisms:

• Comparison with yeast Sld2:

  • Both are essential for DNA replication initiation

  • Both likely undergo CDK-dependent phosphorylation

  • Yeast Sld2 has 11 clustered CDK-phosphorylation motifs, while the exact number in N. crassa sld-2 may differ

• Comparison with S. pombe Drc1:

  • Both function in DNA replication

  • Mutations in conserved regions cause similar growth defects

  • S. pombe Drc1 is known as DNA replication checkpoint protein, suggesting possible additional functions

• Comparison with C. elegans SLD-2:

  • C. elegans SLD-2 has eight CDK consensus sites and is phosphorylated in a cell cycle-dependent manner

  • Phosphorylation regulates interaction with MUS-101 (Dpb11 ortholog)

  • Similar mechanisms likely operate in N. crassa sld-2

• Comparison with vertebrate homologs:

  • RECQL4 has been proposed as a homolog of Sld2 in vertebrates

  • It has weak homology to Sld2 and functions in the initiation of DNA replication

  • The functional conservation between N. crassa sld-2 and RECQL4 remains to be fully established

The table below summarizes key features of sld-2 homologs across different species:

Understanding these evolutionary relationships provides valuable context for interpreting experimental results and designing cross-species studies.

What are the promising approaches for studying sld-2 function in N. crassa moving forward?

Several methodological approaches show particular promise for advancing our understanding of sld-2 function in N. crassa:

• CRISPR/Cas9 gene editing:

  • The recently developed user-friendly CRISPR/Cas9 system for N. crassa offers efficient targeted modification of sld-2

  • Combined targeting approaches (using selectable markers alongside sld-2) can increase mutation efficiency tenfold

  • This enables rapid generation of specific mutations to test functional hypotheses

• Phosphoproteomic analysis:

  • Mass spectrometry-based approaches can identify all phosphorylation sites on sld-2

  • Temporal analysis during the cell cycle can reveal the sequence of phosphorylation events

  • This would help determine if N. crassa sld-2 follows similar regulatory principles as yeast Sld2

• Single-molecule approaches:

  • Techniques like single-molecule FRET could reveal conformational changes upon phosphorylation

  • Super-resolution microscopy might visualize sld-2 localization at individual replication origins

  • These approaches could provide mechanistic insights at unprecedented resolution

• Integration with chromatin studies:

  • Investigating how chromatin context affects sld-2 function at origins

  • Exploring potential links between sld-2 and chromatin remodeling complexes

  • Understanding how epigenetic factors influence sld-2 recruitment and activity

• Systems biology approaches:

  • Mathematical modeling of how limiting amounts of sld-2 affect global replication dynamics

  • Network analysis to position sld-2 within the broader replication initiation complex

  • These approaches could explain how sld-2 abundance affects origin efficiency without changing timing

The combination of these approaches, particularly the integration of CRISPR/Cas9 technology with biochemical and cellular studies, promises to substantially advance our understanding of sld-2 function in N. crassa.

What unresolved questions about sld-2 represent the most important areas for future research?

Several critical questions about N. crassa sld-2 remain unanswered and represent important areas for future investigation:

• Regulatory network:

  • What is the complete set of proteins that interact with sld-2 in N. crassa?

  • Does N. crassa sld-2 function in pathways beyond DNA replication?

  • How is sld-2 expression regulated at the transcriptional and post-transcriptional levels?

• Phosphorylation cascade:

  • Does N. crassa sld-2 employ a similar phosphorylation threshold mechanism as yeast Sld2?

  • What is the identity of the critical phosphorylation site(s) equivalent to Thr84 in yeast?

  • Which kinases and phosphatases regulate sld-2 phosphorylation in vivo?

• Origin selection and efficiency:

  • What determines which origins are most sensitive to sld-2 depletion?

  • How does sld-2 contribute to the decision of which origins fire in each cell cycle?

  • Does sld-2 play a role in adapting replication programs to different growth conditions?

• Connection to genome stability:

  • Does sld-2 play a role in the DNA damage response in N. crassa?

  • How does sld-2 dysfunction contribute to genomic instability?

  • Are there connections between sld-2 and meiotic recombination regulation?

• Evolutionary considerations:

  • What selective pressures have shaped the evolution of sld-2 in filamentous fungi?

  • How do functional differences between sld-2 homologs relate to differences in life cycle and genome organization?

  • Could sld-2 be a target for antifungal development based on fungal-specific features?

Addressing these questions will require integrating multiple experimental approaches and may reveal unexpected functions and regulatory mechanisms of this essential DNA replication factor.