Recombinant Schizosaccharomyces japonicus Flap endonuclease 1 (rad2)
Description
Introduction and Overview
Recombinant Schizosaccharomyces japonicus Flap Endonuclease 1 (Rad2) is a structure-specific nuclease critical for DNA replication, repair, and genomic stability. While Schizosaccharomyces pombe Rad2 has been extensively characterized, limited direct studies exist for S. japonicus Rad2. This review synthesizes findings from homologous systems (primarily S. pombe) to infer its biochemical and functional properties. Rad2 belongs to the XPG/RAD2 endonuclease family and shares evolutionary conservation with human FEN1, playing roles in Okazaki fragment maturation, base excision repair (BER), and replication fork rescue .
Biochemical Characterization
Rad2 was first recombinantly expressed as a GST-fusion protein in Saccharomyces cerevisiae and purified via GST affinity chromatography . Key biochemical properties include:
Enzymatic Activities and Substrate Specificity
Rad2 exhibits dual enzymatic activities:
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5'-Flap Endonuclease Activity: Cleaves 5' overhangs in DNA flap structures, critical for Okazaki fragment processing .
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5'→3' Double-Stranded DNA Exonuclease Activity: Degrades dsDNA, suggesting roles in repair and recombination .
Substrate Preferences:
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Optimal cleavage occurs at branched DNA structures (e.g., 5'-flaps, pseudo-Y substrates).
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Activity is Mg²⁺-dependent and inhibited by secondary structures (e.g., hairpins) .
Role in DNA Repair Mechanisms
Rad2 contributes to multiple DNA repair pathways:
Base Excision Repair (BER)
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Processes intermediates during long-patch BER by excising displaced flaps .
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rad2 mutants in S. pombe exhibit hypersensitivity to alkylating agents (e.g., methyl methanesulfonate), underscoring its role in alkylation damage repair .
Okazaki Fragment Maturation
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Collaborates with DNA polymerase δ and DNA ligase I to resolve RNA primers during lagging-strand synthesis .
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Overexpression of rad2⁺ suppresses temperature-sensitive dna2 mutants, indicating functional redundancy in flap processing .
Double-Strand Break Repair
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Genetic interactions with rad32 (homolog of human MRE11) suggest involvement in homologous recombination repair .
Interaction with Other Proteins
Rad2 forms complexes with replication and repair machinery:
Implications in Genomic Stability and Disease
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Repeat Expansion Prevention: Rad2 resolves secondary structures in DNA flaps (e.g., triplet repeats), preventing mutagenic expansions linked to diseases like Huntington’s .
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Cancer Relevance: Human FEN1 overexpression correlates with microhomology-mediated repair errors and tumorigenesis . While direct links to S. japonicus Rad2 are unexplored, conserved mechanisms suggest analogous roles .
Table 1: Enzymatic Activity of Recombinant Rad2
| Substrate | Cleavage Site | Activity Level | Reference |
|---|---|---|---|
| 5'-Flap DNA | 1 nucleotide 3' of branch point | High | |
| Pseudo-Y DNA | Duplex region adjacent to branch | Moderate | |
| Double-Stranded DNA | 5'→3' exonuclease degradation | Low |
Table 2: Genetic Interactions of rad2⁺
Product Specs
Target Background
STRING: 402676.XP_002172898.1
Q&A
What is Flap Endonuclease 1 (rad2) and what are its key functions in S. japonicus?
Flap Endonuclease 1 (FEN-1) proteins, including the rad2-encoded enzyme in S. japonicus, are nucleases essential for lagging strand DNA synthesis. Based on characterization of the S. pombe homolog, these enzymes possess both 5'-flap endonuclease activity and 5'→3' double-stranded DNA exonuclease activities . The primary functions include:
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Processing of Okazaki fragments during DNA replication
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Removal of 5' flap structures during DNA repair processes
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Participation in long-patch base excision repair pathways
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Maintenance of genome stability, particularly in repetitive DNA regions
The S. japonicus rad2 gene likely encodes a protein with functional similarity to its well-characterized S. pombe counterpart, though with potential adaptations specific to S. japonicus biology.
How does the structure of S. japonicus Rad2 compare to other FEN-1 homologs?
While the specific structure of S. japonicus Rad2 has not been fully characterized in the available literature, comparative analysis with the S. pombe homolog suggests conservation of key structural features:
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A nuclease domain containing catalytic residues essential for both endonuclease and exonuclease activities
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DNA-binding motifs that facilitate substrate recognition
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Potential interaction sites for partner proteins like PCNA
S. japonicus has unique biological characteristics compared to other Schizosaccharomyces species, including striking hyphal growth not observed in S. pombe . These distinctive features may be reflected in subtle structural adaptations of key proteins including Rad2, potentially affecting protein-protein interactions or substrate preferences.
What is the optimal expression system for recombinant S. japonicus Rad2 production?
Based on successful strategies for S. pombe Rad2, several expression systems may be suitable for recombinant S. japonicus Rad2:
For the S. pombe homolog, a GST-Rad2p fusion protein was successfully overexpressed in Saccharomyces cerevisiae and purified to near homogeneity by GST affinity chromatography , suggesting this might be an effective approach for S. japonicus Rad2 as well.
What purification strategy yields the highest enzymatic activity for recombinant S. japonicus Rad2?
A multi-step purification strategy based on successful approaches for related FEN-1 proteins would include:
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Affinity chromatography: GST-fusion tag approach has proven successful for S. pombe Rad2
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Ion exchange chromatography: To remove contaminants based on charge differences
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Size exclusion chromatography: Final polishing step to ensure homogeneity
Critical buffer considerations to maintain enzymatic activity include:
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Inclusion of 5-10 mM MgCl₂ as a cofactor
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Addition of reducing agents (1-5 mM DTT) to maintain cysteine residues
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10-20% glycerol to enhance protein stability
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pH range of 7.5-8.0, optimal for nuclease activity
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Protease inhibitors during initial extraction steps
How can researchers validate the structural integrity of purified recombinant S. japonicus Rad2?
Multiple complementary approaches should be employed to assess proper folding and structural integrity:
| Validation Method | Purpose | Expected Results for Properly Folded Protein |
|---|---|---|
| Enzymatic activity assays | Functional validation | Specific cleavage of 5'-flap structures; exonuclease activity on double-stranded DNA |
| Circular dichroism (CD) spectroscopy | Secondary structure analysis | Profile consistent with mixed α/β structure typical of FEN-1 family |
| Thermal shift assay | Stability assessment | Clear melting transition; enhancement in presence of cofactors (Mg²⁺) |
| Size exclusion chromatography | Oligomeric state | Predominantly monomeric elution profile |
| Limited proteolysis | Structural domain analysis | Discrete digestion pattern reflecting compact domains |
The most definitive validation comes from enzymatic activity assays using defined substrates, as performed for S. pombe Rad2 .
What are the optimal reaction conditions for S. japonicus Rad2 enzymatic assays?
Based on characterization of related FEN-1 proteins including S. pombe Rad2, the following conditions are likely optimal:
| Parameter | Optimal Condition | Notes |
|---|---|---|
| Temperature | 30°C | Matches physiological temperature for S. japonicus growth |
| pH | 7.5-8.0 | Typical optimum for nucleases |
| Divalent metal ion | 5-10 mM MgCl₂ | Essential cofactor; Mn²⁺ may substitute but potentially alter specificity |
| Salt concentration | 50-100 mM NaCl or KCl | Higher concentrations may reduce activity |
| Reducing agent | 1 mM DTT | Maintains cysteine residues in reduced state |
| Buffer | 50 mM Tris-HCl | Provides appropriate pH stability |
| Substrate concentration | 10-100 nM | For initial rate determinations |
The specific nuclease activity of S. pombe Rad2p has been demonstrated using various oligonucleotide structures, with the enzyme showing the ability to incise a 5'-flap and a 5'-pseudo-Y structure one base 3' of the branch point in the duplex region .
How does S. japonicus Rad2 activity differ across various DNA substrate structures?
Expected substrate preferences based on S. pombe Rad2p characterization:
| Substrate Structure | Relative Activity | Cleavage Pattern |
|---|---|---|
| 5'-flap structure | High | One nucleotide into duplex from branch point |
| 5'-pseudo-Y structure | High | One nucleotide into duplex from branch point |
| Double-stranded DNA | Moderate | Progressive 5'→3' exonuclease activity |
| Nicked DNA | Moderate | Exonucleolytic digestion from nick |
| Single-stranded DNA | Low/None | Not a preferred substrate |
| Holliday junctions | Low | Minor activity, if any |
The dual 5'-flap endonuclease and 5'→3' exonuclease activities of S. pombe Rad2p suggest S. japonicus Rad2 would maintain similar substrate preferences, with highest activity on structures mimicking DNA replication and repair intermediates .
What is the effect of divalent metal ions on S. japonicus Rad2 catalytic activity?
As a member of the FEN-1 family, S. japonicus Rad2 likely exhibits strong metal ion dependency:
| Metal Ion | Effect on Activity | Optimal Concentration |
|---|---|---|
| Mg²⁺ | Primary physiological cofactor | 5-10 mM |
| Mn²⁺ | Supports activity, potentially with altered specificity | 0.5-2 mM |
| Ca²⁺ | Likely inhibitory | N/A |
| Zn²⁺ | Low concentrations may be structural; higher concentrations inhibitory | <0.1 mM (structural) |
| EDTA | Complete inhibition | N/A |
Metal ions are essential for catalysis, providing charge neutralization and activating water molecules for nucleophilic attack on the phosphodiester bond. S. pombe Rad2p, like other FEN-1 family members, requires divalent metal ions for both endonuclease and exonuclease activities .
How does S. japonicus Rad2 participate in DNA replication?
S. japonicus Rad2, based on its homology to S. pombe Rad2p and other FEN-1 proteins, likely plays several critical roles in DNA replication:
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Okazaki fragment processing: Removes RNA primers and associated DNA during lagging strand synthesis
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Resolution of secondary structures: Processes aberrant DNA structures that can form during replication of repetitive sequences
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Replication restart: Potential role in processing stalled replication forks, as suggested by the functional relationship between Rad2 and DNA polymerase α
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Prevention of repeat expansion: Studies in S. cerevisiae have shown that defects in FEN-1 homologs can lead to expansions of CAG repeat tracts, suggesting Rad2 may have a similar role in maintaining stability of repetitive sequences
The functional relationship between flap endonuclease Fen1 (Rad2) and DNA polymerase α in S. pombe suggests these enzymes work cooperatively during DNA replication and in response to DNA damage .
What is the specific role of S. japonicus Rad2 in various DNA repair pathways?
Based on functional studies of FEN-1 homologs and S. pombe Rad2p specifically, S. japonicus Rad2 likely participates in multiple DNA repair pathways:
In S. pombe, cells with the swi7-1 mutation (in DNA polymerase α) are hypersensitive to DNA damaging agents including methyl methanesulfonate (MMS), hydroxyurea (HU), and UV, with Fen1 (Rad2) functioning in the same pathway for alkylation damage response .
How does S. japonicus Rad2 contribute to genome stability?
S. japonicus Rad2 likely maintains genome stability through several mechanisms:
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Prevention of replication errors: By properly processing Okazaki fragments, Rad2 prevents accumulation of unligated DNA fragments and potential genome rearrangements
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Maintenance of repetitive sequences: FEN-1 homologs are known to be important for the stability of simple repetitive DNA sequences, with deficiencies leading to repeat expansions
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Resolution of DNA damage: Through its role in multiple DNA repair pathways, Rad2 prevents persistence of DNA lesions that could lead to mutations or chromosomal aberrations
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Prevention of recombination at inappropriate sites: By efficiently processing replication intermediates, Rad2 may reduce opportunities for inappropriate recombination
Studies in S. pombe have shown that deficiencies in the DNA repair pathway involving Fen1 (Rad2) lead to elevated levels of repair foci and increased recombination, indicating genome instability .
How conserved is the rad2 gene across Schizosaccharomyces species?
The rad2 gene shows significant conservation across Schizosaccharomyces species, reflecting its essential role in DNA metabolism:
Despite their evolutionary distance, S. pombe and S. japonicus both maintain rad2 genes with conserved catalytic domains, highlighting the essential nature of this enzyme. The structural and functional conservation of rad2 is particularly noteworthy given that S. japonicus exhibits unique biological characteristics not found in other Schizosaccharomyces species, including hyphal growth .
What functional adaptations might S. japonicus Rad2 have evolved compared to other fission yeast homologs?
S. japonicus has several distinctive biological features that might be associated with functional adaptations in key proteins including Rad2:
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Hyphal growth adaptation: S. japonicus uniquely exhibits striking hyphal growth not seen in other Schizosaccharomyces species , potentially requiring specialized DNA replication and repair mechanisms during morphological transitions
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Sporulation efficiency: Some S. japonicus strains show elevated sporulation even under nitrogen-abundant conditions , which might be associated with alterations in DNA metabolism proteins
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Structural modifications: Potential alterations in protein interaction domains to accommodate the unique cellular environment and partner proteins in S. japonicus
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Regulatory adaptations: Possible differences in expression regulation or post-translational modifications to coordinate with S. japonicus-specific cellular processes
A detailed comparative analysis of S. japonicus Rad2 with its homologs would require experimental characterization of the recombinant protein alongside its S. pombe counterpart.
How does the genomic context of the rad2 gene in S. japonicus compare to other species?
While detailed information about the genomic context of rad2 in S. japonicus is limited in the search results, we can infer potential characteristics based on comparative genomics principles:
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Regulatory elements: The promoter region of rad2 likely contains binding sites for DNA damage-responsive transcription factors, similar to other organisms
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Chromosomal location: The genomic neighborhood may contain functionally related genes involved in DNA replication or repair
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Gene structure: The intron-exon organization might differ between S. japonicus and S. pombe, reflecting their evolutionary divergence
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Associated non-coding RNAs: Potential regulatory ncRNAs might be associated with the rad2 locus, influencing its expression
Comparative analysis of the genomic context across species could provide insights into the evolution of regulatory mechanisms controlling rad2 expression and potential co-regulation with functionally related genes.
How can recombinant S. japonicus Rad2 be applied in synthetic biology and biotechnology?
The enzymatic properties of S. japonicus Rad2 suggest several potential applications:
| Application | Methodology | Potential Advantages |
|---|---|---|
| Structure-specific DNA manipulation | Targeted cleavage of engineered DNA structures | Precise DNA processing at defined structures |
| SNP detection systems | Recognition and cleavage of mismatch-containing flap structures | High specificity for variant detection |
| Synthetic DNA assembly | Processing of intermediates during DNA assembly methods | Enhanced accuracy in assembly processes |
| Nanopore sequencing | Sample preparation for third-generation sequencing | Structure-specific DNA processing |
For such applications, protein engineering approaches could be employed to enhance stability, modify substrate specificity, or create fusion proteins with additional functionalities tailored to specific biotechnological needs.
What insights might structural studies of S. japonicus Rad2 provide for understanding FEN-1 mechanism?
Structural characterization of S. japonicus Rad2 could yield valuable insights:
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Comparative structural analysis: Differences between S. japonicus Rad2 and other FEN-1 homologs might reveal structural adaptations associated with S. japonicus' unique biology
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Substrate recognition mechanisms: Co-crystal structures with DNA substrates could elucidate the molecular basis of structure-specific recognition
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Metal coordination: Detailed understanding of the active site architecture and metal binding sites could clarify the catalytic mechanism
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Conformational dynamics: Analysis of protein dynamics during substrate binding and catalysis could reveal essential mechanistic details
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Species-specific features: Identification of structural elements unique to S. japonicus Rad2 might provide insights into evolutionary adaptations
Methodological approaches would include X-ray crystallography, cryo-electron microscopy, and molecular dynamics simulations to capture both static structures and dynamic properties.
How might S. japonicus Rad2 function differ during yeast versus hyphal growth phases?
S. japonicus uniquely exhibits both yeast and hyphal growth forms , raising interesting questions about Rad2 function during these different morphological states:
| Growth Phase | Potential Rad2 Adaptations | Research Questions |
|---|---|---|
| Yeast phase | Standard replication and repair functions | Is Rad2 activity similar to other yeast FEN-1 homologs? |
| Hyphal growth | Potentially altered regulation or localization | Is Rad2 differentially regulated during the transition to hyphal growth? |
| Transition period | Possible involvement in morphological changes | Does Rad2 participate in DNA metabolism changes during the transition? |
RNA sequencing analysis of S. japonicus during mycelial growth revealed that the expression of more than 2000 genes changed in a statistically significant manner compared to yeast-phase cells , suggesting that DNA metabolism proteins including Rad2 might be differentially regulated during these growth phases.
