Quantitative helicase and polymerase assays to accelerate DNA damage repair targeted drug development

Repairing DNA damage is essential for maintaining cellular health and genomic stability. Mammalian cells therefore rely on multiple repair mechanisms, each activating specific response pathways that recruit defined sets of proteins to initiate and execute repair. Beyond their roles in DNA maintenance, many repair proteins also regulate cell-cycle progression, stress responses, and apoptosis. Proteins involved in DNA damage repair (DDR) pathways have become important therapeutic targets in oncology. Some DDR inhibitors enhance the effectiveness of DNA-damaging cancer treatments such as chemotherapy and radiotherapy, while others induce synthetic lethality when combined with genetic alterations or inhibition of complementary DDR pathways. Among emerging targets, DNA polymerases and helicases offer considerable promise. Helicases unwind complex DNA and RNA structures during replication, recombination, and transcription, and their ATPase activity provides the energy required for this unwinding function. Inhibiting ATPase activity disrupts helicase function, leading to increased DNA damage, cell-cycle arrest, and apoptosis in cancer cells. As a result, small molecules that interfere with ATPase or helicase activities represent compelling therapeutic strategies. To support drug discovery and development efforts, we have established quantitative enzymatic assays that measure the activity of multiple DNA polymerases and helicases. These include assays that selectively assess helicase activity or ATP hydrolysis for targets such as WRN, DHX9, and BLM. Each protocol was optimized to account for dual enzymatic functions and substrate preferences. Together, these assays enable robust evaluation of candidate inhibitors, providing insights into potency, mechanism of action, and target selectivity.