DNA2 variant analysis supports the nuclease activity as a preferred therapeutic target

DNA2 is a combined nuclease and helicase that processes long 5’ flaps and other structures that occur during DNA replication and repair. Current models invoke an essential function of DNA2 in processing stalled replication forks to dampen toxic recombination based restart. As an essential replication stress response factor, DNA2 has been proposed as an anti-cancer drug target with several tool compounds developed. Here we sought to model inhibited DNA2 states using dominant genetics with separation-of-function alleles to identify the optimal mechanisms for DNA2 targeting. We find that expression of DNA2 nuclease-dead alleles exert dominant effects on fitness and DNA damage repair that are dependent on its helicase and RPA binding activities. Furthermore, we find an imperfect link wherein a subset of ALT+ models are hypersensitive to the presence of DNA2-nuclease dead protein. These results were replicated with a nuclease-specific inhibitor of DNA2. Together, our data suggests that DNA2 targeting in cancer should be nuclease focused and will rely on identification of specific biomarker subsets within ALT+ or other tumor cell states.