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Applications: Screen or titrate small molecule inhibitors or antagonists that affect helicase activity of BLM in high
throughput screening (HTS) applications.Counter-screen for compounds that affect the exonuclease activity of BLM.
Background: The BLM helicase, also known as Bloom syndrome protein, is a key enzyme involved in DNA replication and repair (DDR). The BLM helicase is a member of the RecQ family of helicases, which are evolutionarily conserved and found in many organisms, including bacteria, yeast, and humans. It catalyzes the unwinding of duplex DNA with 3' to 5' directionality, driven by the energy generated from ATP hydrolysis. BLM plays a crucial role in maintaining genomic stability by unwinding DNA structures during processes such as DNA replication, recombination, and repair. Mutations in the BLM gene can lead to Bloom syndrome, a rare genetic disorder characterized by growth deficiency, sun-sensitive skin lesions, and an increased risk of cancer. High expression of BLM is found in glioblastoma, and it was found that inhibition of its activity leads to increased susceptibility to treatment with drugs targeting other proteins involved in DDR, such as PARP1 (poly-ADP ribosylation protein 1). The use of BLM inhibitors as part of a combinatory therapeutic approach may open new avenues of treatment in cancer therapy.
Contraindications: The final concentration of DMSO in the reaction should not exceed 1%.Fluorescent compounds that have λex=525nm and detection at λem=592 nm can interfere with the
readouts.
Description: The BLM Helicase Activity Assay Kit is a fluorogenic assay designed for screening and profiling of BLM (Bloom syndrome protein) antagonists/inhibitors by monitoring their inhibitory effects on BLM-catalyzed DNA unwinding. The BLM Helicase Activity Assay Kit comes in a convenient 96-well format, with enough purified recombinant BLM (amino acids 630-1300), ATP, DNA substrate, assay buffer, and additives for 100 reactions. The DNA substrate is conjugated on one strand with the TAMRA (tetramethylrhodamine) fluorophore, and on the other strand with BHQ (Black Hole Quencher) which effectively quenches TAMRA fluorescence due to their proximity within the DNA double strand. BLM unwinding of the DNA probe separates the two strands, releasing TAMRA fluorescence. BLM activity, therefore, results in a proportional increase in fluorescence.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed. Avoid multiple freeze/ thaw cycles!
Uniprot: P54132
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
References: Chen, X., et al., 2021. eLife 10 :e65339.
Wojnicki K., et al., 2023 Cell Death Discovery 9 :157.
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