Abstract
The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.
Highlights
Ble recruitment of the ATR-ATRIP complex to damaged sites (7, 9)
These observations have led to the general idea that ATM and ATR function in parallel pathways where cellular response to replicative stress and UV radiation is attributed to ATR, whereas ATM mediates cellular responses to ionizing radiation (IR)-induced double strand breaks
The association that occurred in untreated cells was not altered by DNA damage, and we were unable to detect a direct interaction between purified proteins
Summary
Ble recruitment of the ATR-ATRIP complex to damaged sites (7, 9). there may be alternative mechanisms by which the ATRATRIP complex can recognize damaged DNA other than direct ATRIPRPA-ssDNA binding (9 –14). ATM primarily initiates cellular responses to double strand breaks and is recruited to sites of damage by the Mre11-Rad50Nbs[1] (MRN) complex (14 –17). Cells from A-T patients are sensitive to IR but not to UV exposure These observations have led to the general idea that ATM and ATR function in parallel pathways where cellular response to replicative stress and UV radiation is attributed to ATR, whereas ATM mediates cellular responses to IR-induced double strand breaks. ATM- and Mre11-deficient cells display defects in ATR-dependent Chk[1] phosphorylation. ATR-ATRIP as well as RPA recruitment to double strand breaks is reduced in ATM- and Mre11deficient cells. ATM and Mre[11] stimulate ATR-dependent Chk[1] phosphorylation by promoting the conversion of DNA damage generated by IR into structures that recruit the ATR-ATRIP complex
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