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Conflict of interest statement
Acknowledgments
Introduction Cellular responses to genotoxic stress in eukaryotic cells are coordinated by members of the phosphoinositide kinase related protein kinase (PIKK) family. Two members of this family, Ataxia–Telangiectasia mutated (ATM) and ATM and Rad 3-related (ATR), are particularly important in regulating α-Naphthoflavone checkpoints in response to DNA damage and replication stress [15], [22]. ATM is mutated in patients with the clinical disorder ataxia–telangiectasia (A–T), a disease characterized by a number of debilitating symptoms, including chromosomal instability and cancer predisposition. ATR mutations are rare because ATR function is essential for cellular viability [6], [11]. Hypomorphic mutations in ATR have been found in patients with Seckel syndrome [21]. The ATM and ATR checkpoint signaling pathways may be significant tumorigenesis barriers [4], [5], [14]. ATM and ATR share significant sequence similarity, phosphorylate many of the same substrates, and have overlapping functional activities. However, they respond to different types of genotoxic stress. ATM primarily is activated in response to double strand breaks, while ATR responds to DNA damage or replication stress that exposes regions of single-stranded (ss) DNA [9]. Consequently, ATM-deficient cells are sensitive to genotoxic agents that generate DNA double strand breaks (DSBs), such as ionizing radiation (IR) and radiomimetic chemicals, but they exhibit normal responses to most other types of DNA damaging agents including ultraviolet (UV) radiation and agents that stall replication forks such as hydroxyurea (HU). In contrast, ATR responds to DNA lesions that can initiate the uncoupling of polymerase and helicase activities at replication forks including UV radiation and HU [7]. A common step that regulates ATM and ATR signaling is localization of these kinases to sites of DNA damage or stalled replication fork. ATM localization is mediated by binding to the Nbs1 protein [13]. Nbs1 is one component of the Mre11–Rad50–Nbs1 (MRN) complex that is critical for double strand break repair. ATR localization is dependent on the ATRIP protein, which serves as a subunit of the kinase [3], [11]. ATRIP can recognize single stranded DNA via an interaction with the ssDNA binding protein RPA [26].
Materials and methods
Results
Discussion In this study, we have tested if ATRIP could confer ATR properties to ATM by fusing the N-terminal ATRIP binding domain of ATR onto full-length ATM. The chimeric ATM⁎ protein has the same ability to bind ATRIP as wild-type ATR. It also has gained the ability to bind RPA-ssDNA and localize to sites of replication stress similarly to wild-type ATR. In addition, ATM⁎ is an active kinase in vitro and is phosphorylated on the activating site S1981 in cells. However, ATM⁎ cannot substitute for either ATR or ATM in cells. Our data indicate that the N-terminus of ATR is sufficient to bind to ATRIP and promote localization of a PIKK kinase to sites of replication stress. However, localization and autophosphorylation of ATM is not sufficient to promote checkpoint signaling. Although ATM⁎ localizes to sites of DNA damage it is not functional. ATRIP binding to ATM⁎ may interfere with Nbs1 binding . Nbs1 binding to ATM promotes both its localization to double strand breaks [13] as well as its activation [17], [18]. Our immunofluorescence studies indicate that ATM⁎ can localize to damage-induced foci likely through its interaction with ATRIP. Although it localizes to irradiation induced foci, the architecture of the checkpoint complexes at the double strand break in the presence of ATM⁎ is likely to be different than with wild-type ATM. This may influence its ability to phosphorylate Chk2. Nbs1 binding may also be important for another step in ATM⁎ activation beyond S1981 phosphorylation and localization. The inability of ATM⁎ to function as ATR may be due to the lack of TopBP1-dependent regulation of the kinase.