Activation of Ataxia Telangiectasia-mutated DNA Damage Checkpoint Signal Transduction Elicited by Herpes Simplex Virus Infection

Abstract

Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection. Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection. Upon DNA damage, eukaryotic cells exhibit a variety of physiological responses, including cell cycle arrest, activation of DNA repair, and apoptosis. Sets of checkpoint proteins that have been conserved with evolution are rapidly induced to prevent replication or segregation of damaged DNA before repair is completed. Related phosphatidylinositol 3-like kinases, ataxia telangiectasia-mutated (ATM) 1The abbreviations used are: ATM, ataxia telangiectacia-mutated; ATR, ATM-Rad3-related; DSB, DNA double strand breaks; FISH, fluorescence in situ hybridization; HFF, human foreskin fibroblast; HSV, herpes simplex virus; hTERT, human telomerase reverse transcriptase gene; HU, hydroxyurea; IR, ionizing radiation; PBS, phosphate-buffered saline; MRN, complex composed of Mre11, Rad50, and Nbs1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; shRNA, short hairpin RNA; GFP, green fluorescent protein; m.o.i., multiplicity of infection; ACV, acyclovir; PAA, phosphonoacetic acid; BrdUrd, bromodeoxyuridine; p.i., postinfection. and ATM-Rad3-related (ATR), respond to a variety of abnormal DNA structures and initiate signaling cascades leading to a DNA damage checkpoint (1Westphal C.H. Curr. Biol. 1997; 7: R789-R792Abstract Full Text Full Text PDF PubMed Google Scholar). For example, ATM responds to the presence of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar). On the other hand, the ATR pathway can be stimulated by hydroxyurea (HU), UV light, and base-damaging agents that interfere with the movement of replication forks (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The ATR pathway also responds to DSBs but more slowly than ATM (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar). A variety of checkpoint proteins have been identified as substrates for ATM and ATR kinases, including the checkpoint kinases Chk1 and Chk2, as well as p53 (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar, 5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar). ATM exists as an inactive dimer in the nucleus but undergoes autophosphorylation at Ser-1981 in response to DSBs and dissociates into active monomers (1Westphal C.H. Curr. Biol. 1997; 7: R789-R792Abstract Full Text Full Text PDF PubMed Google Scholar). ATM phosphorylates Chk2 including Thr-68, followed by Chk2 activation (5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 6Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (874) Google Scholar, 7Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (694) Google Scholar, 8Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar). Chk1 is mainly phosphorylated by ATR in response to UV and HU, leading to a 3–5-fold increase in enzyme activity (5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 6Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (874) Google Scholar, 9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). Both Mre11 and Nbs1 are also targets of ATM and possibly ATR (9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar, 10Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, 11Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar, 12D'Amours D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (718) Google Scholar). The MRN complex consisting of Mre11, Rad50, and Nbs1 has been proposed to facilitate ATM activation (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar, 14Usui T. Ogawa H. Petrini J.H. Mol. Cell. 2001; 7: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 15Williams B.R. Mirzoeva O.K. Morgan W.F. Lin J. Dunnick W. Petrini J.H. Curr. Biol. 2002; 12: 648-653Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) and was recently demonstrated to function upstream of ATM activation as a damage sensor, in addition to acting as an effector of ATM signaling (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar, 16Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar). Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are enveloped double-stranded DNA viruses with genomes of 152 and 155 kbp, respectively (17Roizman B. Knipe D.M. Fields B.N. Knipe D.M. Howley P.M. Griffin D.E. Fields Virology. Fourth Ed. Lippincott Williams & Wilkins, Philadelphia, PA2002: 2399-2459Google Scholar). Upon infection immediate-early gene products are expressed and lead to an ordered cascade of viral early and late gene expression. Viral genome is replicated by viral replication machinery, generating highly branched replication intermediates (18Lehman I.R. Boehmer P.E. J. Biol. Chem. 1999; 274: 28059-28062Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The packaging machinery then cleaves concatemeric DNA to monomeric units, which are packaged into preassembled capsids. In HSV-1, DSBs may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection (19Valyi-Nagy T. Olson S.J. Valyi-Nagy K. Montine T.J. Dermody T.S. Virology. 2000; 278: 309-321Crossref PubMed Scopus (73) Google Scholar, 20Milatovic D. Zhang Y. Olson S.J. Montine K.S. Roberts II, L.J. Morrow J.D. Montine T.J. Dermody T.S. Valyi-Nagy T. J. Neurovirol. 2002; 8: 295-305Crossref PubMed Scopus (47) Google Scholar). DSBs are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization (21Huang K.J. Zemelman B.V. Lehman I.R. J. Biol. Chem. 2002; 277: 21071-21079Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 22Wohlrab F. Chatterjee S. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6432-6436Crossref PubMed Scopus Google Scholar). is of to host cells can monitor HSV infection as DNA that HSV infection a cellular DNA damage response on DNA damage sensor MRN complex and phosphorylated ATM are recruited to viral replication recognizing newly synthesized viral DNAs as abnormal DNA structures. foreskin cells and green cells were and in Dulbecco's modified Eagle's with fetal calf ataxia were by of the human telomerase reverse transcriptase gene H. H. Y. T. S. M. K. J. 2002; PubMed Scopus (30) Google Scholar). a with a in the gene were by of gene as H. H. Y. T. S. M. K. J. 2002; PubMed Scopus (30) Google Scholar) to of cells with the gene not and cells were in with and while 293T cells with ATM or were in with and human cells were used for of human and the were on cells for by UV light, a of HSV in a was to UV a at a of for The and the gene Y. T. A. K. M. PubMed Scopus Google Scholar) were Y. A HSV was by of and DNAs into cells and by 293T cells as Y. T. A. K. M. PubMed Scopus Google Scholar). were on of cells at multiplicity of infection 1 at were with was used at a of was used at a of T. A. M. Y. H. N. T. J. PubMed Scopus Google Scholar). The was with the virus and in for the of infection. gene was by of and were and and and were Cell and were and highly for and were were with phosphate-buffered and with 1 1 including a and for on of proteins were on and to with and with an were as A. M. T. K. Y. S. Y. T. J. 2003; PubMed Scopus Google Scholar). in were with and then with T. A. M. Y. H. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) a on followed by with at The cells were for 1 with in and at with The cells were with the for at then for 1 with or with or and in was as T. A. M. Y. H. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The was used at a of other were at and the at synthesized DNAs were by cells with for 1 to were as and then for with 2 to before and with for For an was in products to a of DNA H. M. H. T. M. K. M. T. J. 1991; PubMed Scopus Google Scholar) were with by and used for of HSV was as and then the were in to in on cells were with in and with a in DNA DNA and and cells were at for then at were at with in for and cells were in and in of Viral or cells were with at an of 1 or and at the by into the and at the was for 1 on and virus were by on cells Y. F. Virology. PubMed Scopus Google Scholar). of 293T silencing ATM, the to ATM were expressed as by 293T cells were with the and cells were in B. of the and of 293T cells be HSV a DNA a cellular DNA damage response was induced upon HSV the phosphorylation of DNA proteins in cells The ATM responds to and pathway can during of the cell has been recently proposed that ATM is present as an inactive and is by autophosphorylation at Ser-1981 DSBs or in the M.B. Nature. 2003; PubMed Scopus Google Scholar). in A and of cell that of the phosphorylated of ATM at Ser-1981 upon and although of ATM infection. The MRN complex consisting of Mre11, Rad50, and Nbs1 has been to as a damage sensor D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (718) Google Scholar, 16Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google ATM activation (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar). Activated ATM also phosphorylates Nbs1 M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, 11Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar). in A and increase in of the of Nbs1 was or with or The of Nbs1 was phosphorylated as by not The of Mre11 HSV the C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar, T.H. C.T. Weitzman M.D. Nature. 2002; PubMed Scopus Google Scholar). In the presence of ATM is known to on Chk2 R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar, Piwnica-Worms H. C.E. 2000; Google Scholar). with also phosphorylation of Chk2 at in and infection A and phosphorylation of p53 at a of ATM was at and on or well with the recently that phosphorylation of p53 at on ATM R.D. J. PubMed Scopus Google Scholar). In phosphorylation of p53 to its and in increase in its 2002; PubMed Scopus Google Scholar, Nat. Rev. Cancer. PubMed Scopus Google Scholar). infection had no effect on of p53 is also with the that the p53 are not in the infection R.D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). data that HSV infection cellular DNA damage In was not with UV-inactivated virus can and into the cells J. PubMed Google but of the early viral and viral DNA replication not were cells were with a replication-defective virus Y. T. A. K. M. PubMed Scopus Google Scholar) at an of the ATM response was although the DNA was expressed In the presence of ACV, the ATM response by the HSV infection was not is into viral DNA with and viral DNA The of viral DNA viral DNAs and elicit the ATM DNA damage response P. R. B. 2002; PubMed Scopus Google Scholar). In PAA, a of the viral DNA to the ATM DNA damage at multiplicity of infection although the gene viral early was expressed However, at the DNA damage response was induced to in the presence of not viral DNA replication and viral DNA be by host damage the that viral DNA activation of the DNA damage response upon HSV but the that viral gene the DNA damage be that not ATM but also ATR kinases Chk2 at and its activity R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar, Piwnica-Worms H. C.E. 2000; Google Scholar). of p53 at is also by kinases A. Lee A. A. Nat. Cell Biol. 2003; PubMed Scopus Google Scholar, K.M. Williams Y. 1999; PubMed Scopus Google Scholar). HSV infection activate the ATM, ATR, or The ATR responds to DNA replication during (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). can also respond to DSBs the but than In to the Chk2 phosphorylation of Chk1 at is known to be mainly by ATR leading to its activation (9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). Chk1 phosphorylation at in and cells phosphorylation was observed in of cells with a well of the replication induced phosphorylation of on the pathway to be in the cells. that replication with types 1 and 2 activation of ATM DNA damage checkpoint signaling than the ATR pathway that responds to replication and infection cellular DNA damage response, the were with HSV Mre11 and Nbs1 the MRN and ATM in Viral DNA replication occurs at discrete sites in nuclei, replication where viral replication proteins cluster and viral DNAs are The viral DNAs have large concatemeric and and structures (18Lehman I.R. Boehmer P.E. J. Biol. Chem. 1999; 274: 28059-28062Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). ATM was recruited to the viral replication The cells were with to of viral or cellular proteins and then to in A and the replication was in sites in the of cells. HSV infection cellular DNA and viral DNA replication A. Chen M. Knipe D.M. Virology. PubMed Scopus Google Scholar). The sites were with the of newly synthesized viral DNA as by and A and the sites of viral DNA proteins were used as for viral replication DNA damage proteins in infection. in in the cells, ATM phosphorylated at Ser-1981 was found to be to and with viral DNAs in the replication the effect of infection on the of the Mre11 and Nbs1 and in of Mre11 and Nbs1 in the as has been J. H. S. A. K. S. T. Tamai K. K. K. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Cerosaletti K.M. Concannon P. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google indicating that the MRN complex is and retained in the damaged Upon Mre11 and Nbs1 proteins to and in the viral replication by the and the of Mre11 and Nbs1 were in the fluorescence to with a indicating that the Mre11 and Nbs1 proteins not but also retained the of newly synthesized viral DNA. The ATM and MRN complex newly synthesized viral DNA in the replication as abnormal DNA structures and to is that the complex function as a of in of viral genome than as a DNA damage and D.E. J. PubMed Scopus Google Scholar) have that Nbs1 with DNA in replication well with DNA by HSV in Nbs1-deficient but ATM-deficient the of ATM and Nbs1 in DNA damage upon HSV phosphorylation of substrates during infection with the was in cells for ATM or Nbs1 In ATM-deficient cells, the of Nbs1 was of Nbs1 phosphorylation of on Chk2 was not of on p53 was not observed and of p53 were as viral replication as was observed in cells These activation of ATM DNA damage pathway by HSV infection. On the other hand, in Nbs1-deficient cells, infection with the to phosphorylation of substrates for ATM activity as ATM Chk2 Thr-68, and p53 although The data that ATM DNA damage were induced by HSV infection in the of a functional MRN complex but that Nbs1 is for activation of ATM DNA damage the in which ATM and MRN complex in the early of the response has been recently that functional MRN is for ATM activation by DSBs and for activation of (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar). of Nbs1 at Viral of ATM DNA of MRN at viral replication is on the ATM in radiation of cells in of Nbs1 to the damaged nuclei, with of of MRN at sites of damage of ATM in response to DSBs A. Lee A. A. Nat. Cell Biol. 2003; PubMed Scopus Google Scholar, O.K. Petrini J.H. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google Scholar). cells were with of Nbs1 to viral replication The of Nbs1 with that for the viral replication the sites of viral DNA These that of the MRN complex to viral replication is of ATM activation. to ATM at replication in the of a functional MRN cells were with infection in of to the sites of viral replication the ATM to be in the of a functional MRN DNA by HSV Viral the effect of ATM activation on HSV DNA replication, of ATM gene or into 293T cells a and cell be that in 293T cells p53 activity is by downstream as are not in of ATM but not ATR not was ATM silencing a in phosphorylation of Nbs1 by infection as by on The 293T cells with ATM were then with at or and the of in that the of was and 293T cells at a multiplicity of infection. the that virus viral also HSV infection at the of that ATM DNA damage signaling is not for HSV DNA replication and virus in the present that the DNA damage signaling is by HSV probably replication during viral genome The MRN complex in a pathway with ATM X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google ATM as a damage sensor, in addition to acting as an effector of ATM signaling C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar). of ATM, Mre11, and Nbs1 proteins to HSV replication that damage newly synthesized viral DNAs as abnormal DNA structures. with an in of large viral genomes that are as abnormal DNA structures by the MRN leading to activation of cell cycle T.H. C.T. Weitzman M.D. Nature. 2002; PubMed Scopus Google Scholar). a ATM damage response signaling is and Chk2, p53, Nbs1, and ATM are phosphorylated C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar, T.H. C.T. Weitzman M.D. Nature. 2002; PubMed Scopus Google Scholar). HSV DNA replication products are to be large with branched structures that be generated by with replication events (18Lehman I.R. Boehmer P.E. J. Biol. Chem. 1999; 274: 28059-28062Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, M.D. M. Cell. Full Text PDF PubMed Scopus Google Scholar). viral DNA is an a of structures are including large to in DNA as well as structures and replication A. J. PubMed Google Scholar). present data the that newly synthesized viral DNA is by cellular DNA damage has been that a in the of viral replication intermediates Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, Boehmer P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). In repair, the MRN with of other the DNA to for DNA and strand the MRN complex be by ATM, Nbs1 is of the MRN complex and is a for ATM phosphorylation (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar, M. EMBO 2003; PubMed Scopus Google Scholar). The of MRN has activity that is to facilitate DNA M. EMBO 2003; PubMed Scopus Google Scholar, A. L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that the recruited MRN complex be in of the viral DNA. be in the phosphorylates Chk2 at and p53 at the ATR targets Chk1 at leading to Chk1 activity (5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 6Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (874) Google Scholar, 9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). not phosphorylation of on Chk1 in the present The ATR responds to DNA replication during (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Our that HSV infection the ATM DNA damage response ATR signaling as is the with of virus replication infection A. M. Zhang L. N. T. Y. H. Y. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (147) Google Scholar). p.i., cells were the but did not exhibit viral replication with to the in cells, suggesting that HSV DNA replication not in cells in which DNA replication has HSV replication fork of DNA replication, ATR DNA damage checkpoint be HSV replication in or with the of an with activity and Virology. 2000; PubMed Scopus Google Scholar). the complex the MRN signaling ATM and ATR C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar, T.H. C.T. Weitzman M.D. Nature. 2002; PubMed Scopus Google Scholar). the complex function in the of p53 by of p53 P. T. M. D. P.E. 2001; PubMed Scopus Google Scholar). to have to cell cycle checkpoint signaling the with replication of HSV not the MRN the of Mre11 and Nbs1 to be infection However, the viral immediate-early has been to with and p53 R.D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The of p53 by prevent p53 downstream signaling and the of and with of HSV infection. the of checkpoint signaling by HSV be of p53 to prevent of for apoptosis. In HSV has been to gene products by the HSV genome as and that downstream of R. Chen Lee H. L. J. 1999; PubMed Google Scholar, M. J. 1999; PubMed Google Scholar, M. J. 2001; PubMed Scopus Google Scholar). HSV host cellular DNA damage and by multiple In no in viral in 293T cells may be