The ability of the radiomimetic anticancer enediyne C-1027 to induce ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR)-independent damage responses was discovered to reside in its unique ability to concurrently generate robust amounts of double-strand breaks (DSBs) and interstrand cross-links (ICLs) in cellular DNA. Furthermore, a single substitution to the chromophore's benzoxazolinate moiety shifted DNA damage to primarily ICLs and an ATR-but not ATM-dependent damage response. In contrast, single substitutions of the chromophore's -amino acid component shifted DNA damage to primarily DSBs, consistent with its induction of conventional ATM-dependent damage responses of the type generated by ionizing radiation and other radiomimetics. Thus, phosphatidylinositol 3-kinase-like protein kinase regulation of DNA damage responses is dictated by the relative proportions of DSBs and ICLs.DNA double-strand break ͉ C-1027 ͉ radioimetic ͉ ATM ͉ ATR C ells use several members of phosphatidylinositol 3-kinaselike protein kinases (PIKKs), including ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), to initiate cell cycle checkpoint responses to DNA damage (1). The induction of double-strand breaks (DSBs) into cellular DNA rapidly triggers ATM activation, which promotes a kinase cascade involving phosphorylation of the signal transducers Chk1 and Chk2 and downstream targets such as p53, all of which contribute to cell cycle arrest (1, 2). Similar to ATM, ATR activates Chk1, p53, and, to a lesser degree, Chk2 to initiate cell cycle checkpoints in response to lesions such as interstrand cross-links (ICLs), which stall DNA replication (2).Although ATM's role in responding to DSBs was primarily based on ionizing radiation (IR) studies, radiomimetic enediynes such as neocarzinostatin, C-1027, and calicheamicin also have provided insight (3). Enediynes are a structurally diverse group of compounds whose chromophores bind to the DNA minor groove and subsequently undergo Bergman cycloaromatization, generating two free radicals (4, 5). These radicals can induce DSBs when hydrogen atoms in close proximity, but on opposite DNA strands (6), are abstracted from deoxyribose.Cellular responses to radiomimetic treatment are similar to IR, which include ATM-dependent activation of p53-Ser-15 and Chk2-Thr-68 (7, 8) and enhanced cell death in cells lacking ATM (5). That responses to DSBs are ATM-dependent regardless of the damaging agent has led to the general conclusion that cells require ATM to activate DNA damage responses to DSBs (5, 6). The sole exception is C-1027, where cells deficient in either ATM or ATR phosphorylate p53-Ser-15 and Chk2-Thr-68 as readily as wild type and are diminished only when both PIKKs are absent (9, 10). Furthermore, C-1027 is similarly toxic to wild-type, ATM-, or ATR-deficient cells, because cell death hypersensitivity occurs only in the absence of both (10).Remarkably, minor modifications of the C-1027 chromophore can alter the PIKK dependence of the DNA damage response (11). ...