Recombinant Drosophila simulans Flap endonuclease 1 (Fen1)
Description
Functional Role in DNA Metabolism
As a member of the RAD2/XPG nuclease family, Drosophila simulans Fen1 performs three key activities:
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5'-flap endonuclease: Removes displaced RNA/DNA flaps during Okazaki fragment maturation .
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5'-3' exonuclease: Excises mismatched nucleotides in base excision repair (BER) .
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Gap endonuclease: Cleaves DNA at single-strand gaps, preventing replication fork collapse .
Structural studies reveal that Fen1 binds flap junctions via a helical arch and cap domain, threading the 5' flap through its active site for precise cleavage . This mechanism prevents genomic instability caused by unprocessed flaps or secondary structures .
3.1. Replication Fork Protection
Drosophila Fen1 collaborates with Wuho (WH) to balance flap and gap endonuclease activities at replication forks. WH enhances flap cleavage efficiency by 40% while suppressing gap activity by 60%, ensuring fork integrity . This interaction mirrors conserved repair mechanisms observed in human cells .
3.2. R-Loop Resolution
Recent studies show Fen1 resolves R-loops (RNA:DNA hybrids) via BER pathways. It cleaves RNA flaps in hybrid structures with 85% efficiency in vitro, coordinating with APE1 exonuclease to prevent transcriptional-replicative conflicts .
3.3. Evolutionary Conservation Analysis
Comparative studies between Drosophila simulans and human Fen1 highlight:
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78% sequence homology in catalytic domains.
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Identical metal ion coordination (Mg²⁺/Zn²⁺) for phosphodiester bond hydrolysis .
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Divergent regulatory regions, suggesting species-specific protein interactions .
Technical Performance Metrics
| Parameter | E. coli-Expressed (CSB-EP008585DMJ) | Baculovirus-Expressed (CSB-BP008585DMJ) |
|---|---|---|
| Purity (SDS-PAGE) | ≥90% | ≥95% |
| Specific Activity | 1,200 U/mg | 2,500 U/mg |
| Thermostability | Active up to 45°C | Active up to 55°C |
| Storage Stability | -80°C for 12 months | -80°C for 18 months |
Data derived from manufacturer specifications and functional assays
Research Limitations & Future Directions
While recombinant Drosophila simulans Fen1 provides insights into conserved DNA repair mechanisms, challenges remain:
Ongoing work focuses on engineering thermostable variants for industrial applications and mapping interaction networks with replication checkpoint proteins .
Product Specs
Target Background
KEGG: dsi:Dsimw501_GD25512
Q&A
What structural features enable Fen1 to recognize and process bifurcated DNA substrates?
Fen1 belongs to the RAD2 nuclease family and employs a "tracking mechanism" to thread single-stranded DNA (ssDNA) flaps through its helical arch, positioning the scissile phosphate at the junction of duplex DNA . Structural studies reveal three conserved domains:
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Helical cap: Binds the 5'-end of the flap.
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Nuclease domain: Contains Mg²⁺-coordinating residues for catalytic activity.
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C-terminal tail: Mediates protein-protein interactions (e.g., with PCNA) .
Methodological Approach
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Crystallography/X-ray diffraction: Resolve Fen1-DNA co-crystals to identify substrate-binding interfaces.
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Fluorescence resonance energy transfer (FRET): Monitor conformational changes during flap threading .
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Electrophoretic mobility shift assays (EMSAs): Compare binding affinities for substrates like 5'-flaps, gaps, or RNA:DNA hybrids .
Table 1: Key Enzymatic Activities of Fen1
| Activity | Substrate | Assay | Role in DNA Metabolism |
|---|---|---|---|
| 5'-Flap endonuclease | Displaced RNA/DNA flaps | Radiolabeled cleavage assays | Okazaki fragment maturation |
| 5'-3' Exonuclease | Nicked/gapped DNA | ExoIII competition assays | Long-patch BER |
| Gap endonuclease | ssDNA gaps (≥4 nt) | Fluorescent primer hydrolysis | Stalled fork rescue |
How is recombinant Drosophila simulans Fen1 expressed and purified for functional studies?
The Drosophila simulans Fen1 gene (385 amino acids; Uniprot ID: [insert]) is codon-optimized for expression in E. coli or insect cell systems.
Stepwise Protocol
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Vector design: Clone the Fen1 ORF into pET-28a(+) with an N-terminal His-tag for metal-affinity purification .
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Expression: Induce with 0.5 mM IPTG at 18°C for 16 hours to minimize inclusion bodies.
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Purification:
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Ni-NTA chromatography (elution: 250 mM imidazole).
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Size-exclusion chromatography (Superdex 200) to remove aggregates.
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Quality control:
Critical Note
Avoid RNase contamination during purification, as Fen1 exhibits RNase H-like activity on RNA:DNA hybrids .
What in vitro assays are used to characterize Fen1’s substrate specificity?
Flap Endonuclease Assay
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Substrate: 5′-FAM-labeled DNA flap (e.g., 5′-GGGTTAGGG-3′ flap annealed to complementary strands).
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Procedure: Incubate 20 nM Fen1 with 100 nM substrate in 20 mM Tris-HCl (pH 7.5), 1 mM DTT, 10 mM MgCl₂. Resolve products via 15% denaturing PAGE .
Exonuclease Activity Assay
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Substrate: 3′-Cy3-labeled nicked DNA (e.g., 5′-GATCGA[Cy3]TACGT-3′).
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Quantification: Monitor fluorescence increase as Fen1 degrades the nick .
Data Interpretation
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Km and kcat: Calculate using Lineweaver-Burk plots. For Drosophila Fen1, reported Km values range from 2–10 nM for 5'-flaps .
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Inhibition assays: Test FEN1 inhibitors (e.g., p-hydroxymercuribenzoate) to confirm metal-dependent activity .
How does Fen1 resolve R-loops during base excision repair (BER)?
R-loops form when RNA hybridizes with DNA, creating a displaced ssDNA strand. Fen1 cleaves the RNA strand within RNA:DNA hybrids via its endonucleolytic activity, facilitated by APE1’s 3′-5′ exonuclease .
Experimental Design
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Substrate design: Synthesize R-loop mimics with a 5′-RNA flap (e.g., rGrArCrU-DNA hybrid).
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Kinetic analysis: Compare cleavage rates of RNA vs. DNA flaps (kcat ≈ 0.5 min⁻¹ for RNA) .
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Immunoprecipitation: Co-express Fen1 and APE1 in HEK293T cells; validate interaction via Western blot .
Contradiction Alert
While yeast Fen1 efficiently processes RNA flaps, human Fen1 exhibits lower activity, suggesting species-specific adaptations . Always include species-matched controls.
What role does Fen1 play in cancer progression, and how can this be modeled experimentally?
Fen1 overexpression in oral squamous cell carcinoma (OSCC) correlates with immunosuppressive phenotypes (e.g., PD-L1 upregulation) and tumor growth .
Methodological Workflow
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Knockdown models: Transfect OSCC cells with siFEN1; assess proliferation (BrdU assay) and DNA damage (γH2AX foci) .
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Multiplex IHC: Co-stain OSCC biopsies for Fen1, HLA-DR, and PD-L1 to quantify immune evasion .
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In vivo xenografts: Inject Fen1-knockdown cells into NSG mice; measure tumor volume and T-cell infiltration .
Table 2: Fen1-Dependent Pathways in OSCC
| Pathway | Regulatory Mechanism | Functional Outcome |
|---|---|---|
| IFN-γ/JAK/STAT1 | Fen1 knockdown → STAT1 inhibition | PD-L1 downregulation |
| DNA damage response | Fen1 deficiency → γH2AX accumulation | Replication stress-induced apoptosis |
How do post-translational modifications regulate Fen1’s activity in vivo?
Fen1 undergoes phosphorylation (Ser-187), acetylation (Lys-254), and methylation, which modulate its localization and protein interactions .
Phosphorylation Analysis
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Mass spectrometry: Identify modification sites in Fen1 immunoprecipitated from S-phase cells.
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Functional impact: Mutate Ser-187 to Ala (non-phosphorylatable) or Asp (phosphomimetic); test nuclear import using GFP-tagged constructs .
Case Study
Phosphorylation by CDK1 during mitosis relocalizes Fen1 to the cytoplasm, limiting its role in replication and forcing reliance on alternative repair pathways .
