Ataxia Telangiectasia Mutated (ATM) Signaling Network Is Modulated by a Novel Poly(ADP-ribose)-dependent Pathway in the Early Response to DNA-damaging Agents

Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling. Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling. Double-strand breaks (DSB) 5The abbreviations used are: DSB, DNA double-strand break; ATM, ataxia telangiectasia mutated; MRN, Mre11/Rad50/NBS1; SMC1, structural maintenance of chromosomes 1; IR, ionizing radiation; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; PAR, poly(ADP-ribose); PARP, poly(ADP-ribose) polymerase; PARG, poly(ADP-ribose) glycohydrolase; DNA-PK, DNA-dependent protein kinase; ATR, A-T and RAD-3-related; MEF, mouse embryonic fibroblast; A-T, ataxia-telangiectasia; Gy, Gray; GST, glutathione S-transferase. are potentially the most cytotoxic form of DNA damage in human cells because they lead to genomic rearrangements, cancer predisposition, and perhaps cell death if unrepaired or repaired incorrectly (1.O'Driscoll M. Jeggo P.A. Nat. Rev. Genet. 2006; 7: 45-54Crossref PubMed Scopus (463) Google Scholar). Consequently, the DNA damage response involves parallel modulation of redundant signaling pathways leading to lesion detection, processing, and repair. Ataxia telangiectasia mutated (ATM) is a DNA damage-responding kinase that is rapidly activated after the induction of DSB (2.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). Within minutes of DNA damage induction, ATM is recruited and activated in the vicinity of DSBs, where it induces the phosphorylation of a number of proteins required for DNA damage response and repair, including proteins of MRN (Mre11/Rad50/NBS1) complex, p53, SMC1 and histone variant H2AX (3.Lavin M.F. Birrell G. Chen P. Kozlov S. Scott S. Gueven N. Mutat. Res. 2005; 569: 123-132Crossref PubMed Scopus (170) Google Scholar). However, the detailed mechanisms of how ATM is activated and regulates its downstream effectors are not fully understood. Although ATM activation is mainly associated with DSB formation as part of the damage detection mechanism following ionizing radiation (IR), several studies indicate that the signaling kinase ATM is also activated in response to the environmental carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (4.Adamson A.W. Kim W.J. Shangary S. Baskaran R. Brown K.D. J. Biol. Chem. 2002; 277: 38222-38229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5.Beardsley D.I. Kim W.J. Brown K.D. Mol. Pharmacol. 2005; 68: 1049-1060Crossref PubMed Scopus (16) Google Scholar, 6.Stojic L. Cejka P. Jiricny J. Cell Cycle. 2005; 4: 473-477Crossref PubMed Scopus (43) Google Scholar). Poly(ADP-ribose) polymerases (PARPs) are also constitutive factors of the DNA damage surveillance network, acting through a DNA break sensor function (7.Bouchard V.J. Rouleau M. Poirier G.G. Exp. Hematol. 2003; 31: 446-454Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Several observations indicate that poly(ADP-ribosyl)ation also plays an early role in DSB signaling and repair pathways (8.Bryant H.E. Schultz N. Thomas H.D. Parker K.M. Flower D. Lopez E. Kyle S. Meuth M. Curtin N.J. Helleday T. Nature. 2005; 434: 913-917Crossref PubMed Scopus (3706) Google Scholar, 9.Farmer H. McCabe N. Lord C.J. Tutt A.N. Johnson D.A. Richardson T.B. Santarosa M. Dillon K.J. Hickson I. Knights C. Martin N.M. Jackson S.P. Smith G.C. Ashworth A. Nature. 2005; 434: 917-921Crossref PubMed Scopus (4751) Google Scholar, 10.McCabe N. Turner N.C. Lord C.J. Kluzek K. Bialkowska A. Swift S. Giavara S. O'Connor M.J. Tutt A.N. Zdzienicka M.Z. Smith G.C.M. Ashworth A. Cancer Res. 2006; 66: 8109-8115Crossref PubMed Scopus (1029) Google Scholar, 11.Andrabi S.A. Kim N.S. Yu S.W. Wang H. Koh D.W. Sasaki M. Klaus J.A. Otsuka T. Zhang Z. Koehler R.C. Hurn P.D. Poirier G.G. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. 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DNA PubMed Scopus Google the as to the phosphorylation cascade initiated by the ATM protein we how synthesis the early DNA damage signaling that synthesis in cells with the PARP inhibitors is associated with phosphorylation of p53, and SMC1 after DNA damage by with and phosphorylation and of and phosphorylation of SMC1 and histone H2AX also in mouse embryonic we demonstrate that with ATM, that interaction is by and that it functional Cell human cells in with and and A-T as as mouse embryonic and L. H. D. M. PubMed Scopus Google in with and the cells and A-T cells used and in with and and and A-T cells to and and cell the as S. Kastan M.B. PubMed Scopus Google Scholar). of PARP-1 with an in and inhibitors and on for and to and and a with with the of protein with and to to with with the and with for with the the in of and cell or by and with PARP-1 SMC1 SMC1 H2AX and the to of and in in we a of as Kozlov S. Scott S. M. K. D. S.P. M.F. Nat. Genet. PubMed Scopus Google Scholar). 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Turner N.C. Lord C.J. Kluzek K. Bialkowska A. Swift S. Giavara S. O'Connor M.J. Tutt A.N. Zdzienicka M.Z. Smith G.C.M. Ashworth A. Cancer Res. 2006; 66: 8109-8115Crossref PubMed Scopus (1029) Google Scholar, J. M. A. G. Mol. Cell Biol. PubMed Scopus Google Scholar). also that the of A-T cells to the of PARP-1 function is to the activation of This is by the and the of the PARP-1 that of PARP function by in cells leads to the of an important signaling of the DNA damage response that is to the cell in cells ATM. in the of DNA kinase ATM is for its activation following (2.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). ATM also to the kinase for the phosphorylation of on DNA damage (4.Adamson A.W. Kim W.J. Shangary S. Baskaran R. Brown K.D. J. Biol. Chem. 2002; 277: 38222-38229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). it that SMC1, and in an of DNA strand to the formation of DNA and by radiation (4.Adamson A.W. 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M.F. Nat. Genet. PubMed Scopus Google Scholar). the that ATM with through its a functional for between synthesis and ATM demonstrate the of ATM to PAR, to the and studies used in a and that several DNA damage proteins H.E. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. H.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that ATM and ATM in that the of in the H.E. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. C. M. J. 2005; PubMed Scopus Google Scholar, J. Poirier G.G. J. 2003; PubMed Scopus Google Scholar). to are required for we of the of the ATM in the of the by that of the or the the of to with and the mechanism by the ATM we used a to of the of ATM protein kinase in cells M. P. S. Nat. Cell Biol. 2003; PubMed Scopus Google Scholar, Wang S. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the ATM ATM by Although cells of ATM, they are for formation not for the of DSB DNA damage including the of where important by to ATM formation the the ATM an on ATM downstream the phosphorylation of on with cells with ATM a of of phosphorylation in cells with ATM with that ATM is activated following and that of the ATM the of ATM DNA damage of phosphorylation by after with the ATM is to of ATM. to not to required for the activation of ATM, to an important role in activated ATM to the of DNA where it including p53, SMC1, and H2AX. observations that the DNA damage response in cells with synthesis a ATM to of DNA that activation of the DNA damage response we the that PARP-1 might with the ATM signaling in interaction between PARP-1 and ATM by of PARP-1 protein by detection of ATM also PARP-1 following and the of poly(ADP-ribosyl)ation a key of MRN complex, also in the and is in a in cells demonstrate that ATM and is to by a DNA damage response pathways are for the maintenance of genomic and cells an signaling that the detection of DNA by leading to of by in the activation of checkpoint pathways that cell cycle to repair. in the PARP poly(ADP-ribosyl)ation is a of early DNA damage signaling suggests that of poly(ADP-ribosyl)ation metabolism by or of PARP-1 cell in with where is compromised (8.Bryant H.E. Schultz N. Thomas H.D. Parker K.M. Flower D. Lopez E. Kyle S. Meuth M. Curtin N.J. Helleday T. Nature. 2005; 434: 913-917Crossref PubMed Scopus (3706) Google Scholar, 9.Farmer H. McCabe N. Lord C.J. Tutt A.N. Johnson D.A. Richardson T.B. Santarosa M. Dillon K.J. Hickson I. Knights C. Martin N.M. Jackson S.P. Smith G.C. Ashworth A. Nature. 2005; 434: 917-921Crossref PubMed Scopus (4751) Google Scholar, 10.McCabe N. Turner N.C. Lord C.J. Kluzek K. Bialkowska A. Swift S. Giavara S. O'Connor M.J. Tutt A.N. Zdzienicka M.Z. Smith G.C.M. Ashworth A. 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Res. 2006; PubMed Scopus Google Scholar). in the of used in not in cells the that synthesis is in a functional interplay with the ATM signaling that PARP inhibitors used for of ATM, through of strand breaks that the to to DSB N. Turner N.C. Lord C.J. Kluzek K. Bialkowska A. Swift S. Giavara S. O'Connor M.J. Tutt A.N. Zdzienicka M.Z. Smith G.C.M. Ashworth A. Cancer Res. 2006; 66: 8109-8115Crossref PubMed Scopus (1029) Google Scholar, H.E. Helleday T. Res. 2006; PubMed Scopus Google Scholar). in study we the of PARP inhibitors on the early signaling that ATM is activated by DNA by or Although not to ATM, several of indicate that the phosphorylation of p53, SMC1, and H2AX is by the kinase ATM in response to DNA damage by and (2.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar, K. K. E. Kastan M.B. PubMed Scopus Google Scholar). that the and formation a unique signal that or in activation of the ATM signaling that ATM is not activated by to DNA damage by a signal by a structural of chromatin (2.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar, K. K. E. Kastan M.B. PubMed Scopus Google Scholar). to PARP-1 might as a sensor and a DNA damage response through modulation of the ATM signaling the to a function for activated ATM of DNA Consequently, we demonstrate that in the of phosphorylation of p53, SMC1, and H2AX is in cells with and and the cells a in phosphorylation of ATM after or is the in a cell deficient in ATM and not that might for the phosphorylation of and SMC1 in and However, it is that the early response because the that following or is and with and is to instantly after DNA damage in a that is to of DSB A. T. Cancer 2005; PubMed Scopus Google Scholar). are with studies that PARP-1 to DSB by L. C. M. K.M. J. G. 2003; PubMed Scopus Google and with in response to S. J. Wang T. H. E. Chen Mol. Cell 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). However, we a and a of in cells in response to a in the DNA damage repair we are the first to demonstrate and a interaction between ATM and of ATM and ATM and that ATM to PARP-1 is to the ATM and ATM we that ATM in through in critical of ATM and ATM for and of ATM in cells the formation of ATM not of DSB repair in cells with MNNG, a on formation the and the interaction the of ATM to DNA breaks and the phosphorylation of in response to DNA damage it that ATM is to if it is not DNA breaks R. C.J. Kastan M.B. PubMed Scopus Google we a of phosphorylation in cells with the ATM Consequently, the of ATM, as a of the DNA damage to through a as to how might the functional of its as following and the the of ATM with the is required for ATM on chromatin and ATM to of DNA damage J. M. D.A. S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the ATM is highly and its is by the that in A-T a between and ATM that ATM and in a and the that is in the modulation of the DNA damage response through interaction with signaling physical between ATM and might the functional of PARP in it that ATM in S.P. DNA PubMed Scopus Google Scholar). that and that ATM kinase Although it ATM in it is to a where the of on proteins or on PARP-1 the phosphorylation of ATM downstream of DNA damage. This in the induction of a DNA damage response upon interaction PARP inhibitors with the early DNA damage-induced phosphorylation of ATM downstream is that synthesis a and mechanism that to the of ATM protein kinase in cells with DNA-damaging that the phosphorylation of ATM is in the of suggests that redundant pathways to the of ATM to of studies by and PubMed Scopus Google an important role for the MRN as a DNA damage sensor to the of ATM However, also a unique between ATM and poly(ADP-ribosyl)ation in the of DNA damage signaling. of poly(ADP-ribosyl)ation as a critical in an important to in studies on that are by DNA damage and cancer Rouleau and Poirier for critical of also for with the