mec-3 Antibody
Description
Mec3 Protein Function and Antibody Applications
The Mec3 protein forms a heterotrimeric complex with Rad17 and Ddc1, critical for DNA damage checkpoint activation across cell-cycle phases . Key functions include:
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G1/S and Intra-S Checkpoint Activation: Mec3 facilitates Rad53 phosphorylation to delay cell-cycle progression in response to DNA damage (e.g., UV or alkylating agents) .
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Meiotic Chromosome Assembly: Mec3 localizes to meiotic chromosomes in a Rad24-dependent manner, promoting ZMM (Zip1-Zip2-Zip3-Msh4-Msh5) protein assembly for crossover formation .
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Genome Stability: Deletion of MEC3 increases gross chromosomal rearrangement (GCR) rates, particularly in sgs1 and rrm3 mutants defective in helicase activity .
DNA Damage Response Studies
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Dominant-Negative Mutant Analysis: A mec3-dn mutant (lexA-9MYC-MEC3 fusion) disrupts G1/S checkpoint activation but preserves G2/M checkpoint function, revealing phase-specific thresholds for DNA damage responses .
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Immunofluorescence Localization: Anti-Mec3 antibodies detect punctate foci on meiotic chromosomes, independent of ZMM proteins like Zip3 or Msh5 .
Genomic Instability Metrics
The table below summarizes GCR rates in yeast mutants, highlighting MEC3's role in suppressing translocations and de novo telomere additions :
| Genotype | GCR Rate (×10⁻⁹) | Translocation Frequency | Telomere Addition Frequency |
|---|---|---|---|
| Wild Type | 3,615 | 0.6% | <0.2% |
| mec3Δ | 5,569 | 3.4% | <1.7% |
| rrm3 sgs1 mec3Δ | 5,579 | 1,456% | 242% |
Comparative Insights from Related Systems
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C. elegans mec-3 Gene: Though not directly related to yeast Mec3, the C. elegans mec-3 gene encodes a LIM homeodomain protein essential for touch receptor differentiation. Its regulation involves cooperative binding with UNC-86 to activate target genes like mec-4 and mec-7 .
Limitations and Future Directions
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Antibody Specificity: Current anti-Mec3 antibodies are primarily research-grade, with limited commercial availability or validation in non-yeast systems.
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Therapeutic Potential: Unlike monoclonal antibodies targeting human proteins (e.g., TNF-α or HER2) , Mec3 antibodies remain confined to basic research in model organisms.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
Basic Research Questions
What experimental models are optimal for investigating MEC-3’s role in DNA damage checkpoints?
The budding yeast (Saccharomyces cerevisiae) is the primary model for studying MEC-3 due to its conserved DNA damage response pathways. Key methodologies include:
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Rad53 phosphorylation assays: Monitor checkpoint activation via Western blot to detect Rad53 mobility shifts after UV or 4-nitroquinoline-1-oxide (4NQO) treatment .
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Cell cycle synchronization: Use α-factor (G1 arrest) or nocodazole (G2/M arrest) to study phase-specific checkpoint functions .
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Flow cytometry: Track DNA content changes (e.g., 1C to 2C transitions) to assess S-phase progression delays under methyl-methane-sulfonate (MMS) stress .
How do researchers validate MEC-3 interactions within the Rad17 complex?
Co-immunoprecipitation (Co-IP) is critical for confirming interactions between MEC-3, Rad17, and Ddc1. Key steps:
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Use epitope-tagged constructs (e.g., 9MYC-MEC3) to isolate complexes from synchronized cells.
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Perform reciprocal IPs in G1 and G2 phases to assess checkpoint-dependent association dynamics .
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Validate functional relevance by testing mutant strains (e.g., mec3-dn) for defective complex formation .
What assays distinguish MEC-3’s role in G1/S vs. G2/M checkpoints?
Advanced Research Questions
How can dominant-negative MEC-3 mutants dissect checkpoint-specific functions?
The mec3-dn mutant (e.g., lexA-9MYC-MEC3) disrupts G1/S and intra-S checkpoints but preserves G2/M function . Experimental design considerations:
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Conditional expression: Use inducible promoters to study temporal effects.
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Phenotypic comparison: Contrast mec3-dn with full knockouts (mec3Δ) to identify residual checkpoint capacities.
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Sensitivity assays: Expose mutants to genotoxic agents (UV, MMS) in phase-specific arrests to quantify survival rates .
What explains partial Rad53 phosphorylation in MEC-3 mutants during intra-S checkpoint activation?
Intermediate Rad53 phosphorylation in mec3-dn strains suggests:
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Alternative signaling pathways: Redundant kinases (e.g., Chk1) may partially compensate.
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Threshold effects: Suboptimal MEC-3 activity might permit limited signal transduction.
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Experimental validation: Combine mec3-dn with rad53Δ to test synthetic lethality under replication stress .
How to map genetic interactions between MEC-3 and other checkpoint genes?
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Synthetic lethality screens: Pair mec3Δ with deletions in RAD24, DDC1, or MEC1 to identify epistatic relationships.
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Quantitative phenotyping: Measure colony survival or mutation rates under combinatorial DNA damage conditions.
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High-throughput microscopy: Track checkpoint adaptation in double mutants using fluorescent cell cycle reporters .
Key Methodological Insights
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Dominant-negative mutants: Tools like mec3-dn reveal phase-specific checkpoint dependencies, highlighting MEC-3’s modular role in signal transduction .
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Rad53 as a readout: Its phosphorylation status serves as a reliable biomarker for checkpoint integrity across cell cycle phases .
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Context-dependent sensitivity: mec3-dn cells show UV sensitivity in G1 but not G2, underscoring the G2/M checkpoint’s independence from MEC-3 .
