One major conserved DNA repair enzyme is the

One major conserved DNA repair enzyme is the DNA2 helicase/nuclease (DNA2). Complete inactivation of either the helicase or nuclease activity of DNA2 in cells from a wide range of organisms, including yeast and humans, induces retinoic acid receptor arrest and cell death (Budd and Campbell, 1995; Kang et al., 2000; Kim et al., 2005; Duxin et al., 2009, 2012; Wawrousek et al., 2010; Karanja et al., 2012; Lin et al., 2013). Disruption of DNA2 has been associated with human disease. A splice-site mutation that causes decreased levels of human DNA2 gives rise to Seckel syndrome, a primordial dwarfism syndrome. Other mutations are linked to breast cancers (Strauss et al., 2014). Interestingly, the DNA2-deficient Seckel cells show markers of senescence, where cells are viable but cease to proliferate (Shaheen et al., 2014). DNA2 plays three key roles that allow the cancer cells to resist the intrinsic and extrinsic DNA replication stresses induced by chemotherapy or RT (radiotherapy): flap removal during DNA replication, double-strand break (DSB) resection during repair, and stabilization and restart of reversed replication forks (Wanrooij and Burgers, 2015). During replication, DNA2 removes the long 5′ RNA/DNA “flaps” that arise during Okazaki fragment processing in difficult-to-replicate genomic regions (Bae et al., 2001; Budd and Campbell, 1997; Kang et al., 2010; Kao et al., 2004a, b; Masuda-Sasa et al., 2008; Stewart et al., 2008, 2010). In yeast, ScDNA2 is probably the major nuclease for RNA primer removal during Okazaki fragment maturation, in collaboration with flap endonuclease 1 (FEN1 or Rad27) (Bae et al., 2001; Budd and Campbell, 1997). For DSB repair, DNA2 acts in one of the two major DSB resection pathways. DNA2 acts with the Bloom Syndrome (BLM) helicase or Werner Syndrome (WRN) helicase in DSB end resection at a critical early step after licensing by limited cleavage by the MRN/CtIP complex (Budd et al., 2005; Imamura and Campbell, 2003; Zhu et al., 2008; Budd and Campbell, 2009; Niu et al., 2009; Cejka et al., 2010; Nimonkar et al., 2011; Symington and Gautier, 2011; Sturzenegger et al., 2014; Liao et al., 2008, 2011). BLM (or WRN), moving on the 3′ terminated strand, unwinds the duplex end to create a “fork”; DNA2 acts as a nuclease on the complementary strand and degrades the 5′ end to produce 3′ ssDNA tails for strand invasion during homology-directed repair (HDR) and S phase checkpoint activation. This resection activity functions in parallel to and independently of resection by Exonuclease 1 (EXO1), downstream of MRE11 (Shibata et al., 2014). At stalled replication forks, DNA2 acts to stabilize, repair and restart forks to allow completion of replication (Hu et al., 2012; Karanja et al., 2014; Thangavel et al., 2015; Weitao et al., 2003a, b). DNA2 also acts in signaling, as both an activator and a target of checkpoint kinases. For instance, DNA2 is required to directly activate the yeast master signaling kinase ATR (Kumar and Burgers, 2013). Furthermore, DNA2 is a target of checkpoint effector kinase Rad53/Chk1/2, and is required to regulate potentially deleterious fork reversal and template switching during replication fork stalling in yeast and humans (Hu et al., 2012; Lai and Foiani, 2012; Thangavel et al., 2015). DNA2 can also play a negative role when the RAD51, BRCA1, BRCA2 and the FA/BRCA (Fanconi anemia/Breast cancer) pathway is impaired (Hashimoto et al., 2010; Schlacher et al., 2011, 2012; Petermann et al., 2010). Like MRE11, which functions upstream of DNA2, DNA2 is involved in the excessive resection seen in cells deficient in fork protection (Karanja et al., 2014; Wang et al., 2015; Higgs et al., 2015). Thus, DNA2 must be highly regulated to protect genome stability. In this work, we describe a potent inhibitor of these DNA2 DNA replication and repair activities that sensitizes cancer cells to chemotherapies. Mechanistically, DNA2 is well studied, but nevertheless poorly defined. Biochemical and genetic experiments have demonstrated an intricate interaction between the nuclease and helicase (Bae et al., 2001; Budd and Campbell, 2000, 2009; Kao et al., 2004a, b; Levikova et al., 2013). Furthermore, biochemical studies indicate that there is a major DNA binding site interacting at the junction of the flap and downstream duplex DNA that is required for both the nuclease and helicase activities (Stewart et al., 2010). Which motifs in the DNA2 protein govern this major binding site have been elusive, but the inhibitor work we report here clarifies these relationships and both provides functional support and is in turn supported by the recently published X-ray crystal structure of murine DNA2 (Zhou et al., 2015). The conclusions of our mechanistic studies using the inhibitor correlate with the multi-domain interaction of DNA2 with DNA revealed in the X-ray crystal structure of murine DNA2 (Zhou et al., 2015), and our mutational studies of inhibitor susceptibility specifically provide functional support for the model proposed for the role of helicase domain DNA binding contacts in nuclease activation in that study (Zhou et al., 2015).