An ATR- and Chk1-Dependent S Checkpoint Inhibits Replicon Initiation following UVC-Induced DNA Damage

ABSTRACT Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpoint mechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC (1 J/m2). Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolished the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuated UVC-induced Chk1 phosphorylation and reversed the UVC-induced inhibition of replicon initiation, as did overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11.

[1]  S. Fedoroff,et al.  Banding in human chromosomes treated with trypsin. , 1972, Nature: New biology.

[2]  B. Strauss,et al.  Sites of inhibition of in vitro DNA synthesis in carcinogen- and UV-treated Φ174 DNA , 1979, Nature.

[3]  R. Painter,et al.  Radiosensitivity in ataxia-telangiectasia: a new explanation. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[4]  W. Kaufmann,et al.  Ultraviolet radiation inhibits replicon initiation in S phase human cells. , 1980, Biochimica et biophysica acta.

[5]  S. Rabkin,et al.  Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[6]  W. Kaufmann,et al.  Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts. , 1981, Journal of molecular biology.

[7]  R. Painter Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light. , 1985, Mutation research.

[8]  W. Kaufmann,et al.  DNA repair and replication in human fibroblasts treated with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene . , 1985, Biochimica et biophysica acta.

[9]  M. Sasaki,et al.  Enhanced expression of X-ray- and UV-induced chromosome aberrations by cytosine arabinoside in ataxia telangiectasia cells. , 1986, Mutation research.

[10]  W. Kaufmann,et al.  Defective postreplication repair in xeroderma pigmentosum variant fibroblasts. , 1990, Cancer research.

[11]  W. Kaufmann,et al.  DNA repair endonuclease activity during synchronous growth of diploid human fibroblasts. , 1990, Mutation research.

[12]  M. Karin,et al.  The mammalian ultraviolet response is triggered by activation of src tyrosine kinases , 1992, Cell.

[13]  W. Kaufmann,et al.  G1 arrest and cell-cycle-dependent clastogenesis in UV-irradiated human fibroblasts. , 1994, Mutation research.

[14]  M. Karin,et al.  JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain , 1994, Cell.

[15]  R. Paules,et al.  Attenuation of G2 checkpoint function precedes human cell immortalization. , 1995, Cancer research.

[16]  A. Kraft,et al.  jun-NH2-terminal kinase activation mediated by UV-induced DNA lesions in melanoma and fibroblast cells. , 1995, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[17]  R. Paules,et al.  DNA damage and cell cycle checkpoints , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[18]  Stephen J. Elledge,et al.  Cell Cycle Checkpoints: Preventing an Identity Crisis , 1996, Science.

[19]  B. Nelms,et al.  hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks , 1997, Molecular and cellular biology.

[20]  Amanda G Paulovich,et al.  When Checkpoints Fail , 1997, Cell.

[21]  W. Kaufmann,et al.  p53-dependent signaling sustains DNA replication and enhances clonogenic survival in 254 nm ultraviolet-irradiated human fibroblasts. , 1998, Cancer research.

[22]  Y Taya,et al.  Enhanced phosphorylation of p53 by ATM in response to DNA damage. , 1998, Science.

[23]  A. Carr,et al.  Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  John R Yates,et al.  The hMre11/hRad50 Protein Complex and Nijmegen Breakage Syndrome: Linkage of Double-Strand Break Repair to the Cellular DNA Damage Response , 1998, Cell.

[25]  Y Taya,et al.  Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. , 1998, Science.

[26]  W. Kaufmann,et al.  DNA signals for G2 checkpoint response in diploid human fibroblasts. , 1998, Mutation research.

[27]  V. Golubovskaya,et al.  Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein , 1998, Oncogene.

[28]  J. Diffley,et al.  A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication , 1998, Nature.

[29]  S. Schreiber,et al.  Overexpression of a kinase‐inactive ATR protein causes sensitivity to DNA‐damaging agents and defects in cell cycle checkpoints , 1998, The EMBO journal.

[30]  M. Lagally,et al.  In situ visualization of DNA double-strand break repair in human fibroblasts. , 1998, Science.

[31]  J. Sarkaria,et al.  Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. , 1999, Cancer research.

[32]  Xu-Rong Jiang,et al.  Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype , 1999, Nature Genetics.

[33]  S. Jackson,et al.  The ataxia-telangiectasia related protein ATR mediates DNA-dependent phosphorylation of p53 , 1999, Oncogene.

[34]  M. White,et al.  Absence of cancer–associated changes in human fibroblasts immortalized with telomerase , 1999, Nature Genetics.

[35]  B. Price,et al.  Caffeine inhibits the checkpoint kinase ATM , 1999, Current Biology.

[36]  S. Elledge,et al.  Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. , 1999, Science.

[37]  Y Taya,et al.  A role for ATR in the DNA damage-induced phosphorylation of p53. , 1999, Genes & development.

[38]  J. Sarkaria,et al.  The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. , 2000, Cancer research.

[39]  N. Mailand,et al.  Rapid destruction of human Cdc25A in response to DNA damage. , 2000, Science.

[40]  J. Petrini The Mre11 complex and ATM: collaborating to navigate S phase. , 2000, Current opinion in cell biology.

[41]  A. Kumagai,et al.  Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. , 2000, Genes & development.

[42]  P. May,et al.  Cell cycle control and cancer. , 2000, Pathologie-biologie.

[43]  J. Shay,et al.  The establishment of telomerase-immortalized cell lines representing human chromosome instability syndromes. , 2000, Human molecular genetics.

[44]  Edward A. Sausville,et al.  The Chk1 Protein Kinase and the Cdc25C Regulatory Pathways Are Targets of the Anticancer Agent UCN-01* , 2000, The Journal of Biological Chemistry.

[45]  Bo Xu,et al.  ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway , 2000, Nature.

[46]  Y. Shiloh,et al.  Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products , 2000, Nature.

[47]  D. Baltimore,et al.  ATR disruption leads to chromosomal fragmentation and early embryonic lethality. , 2000, Genes & development.

[48]  K. Cimprich,et al.  Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint , 2000, Current Biology.

[49]  A. Carr,et al.  Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice , 2000, Current Biology.

[50]  S. Elledge,et al.  Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. , 2000, Genes & development.

[51]  D. Livingston,et al.  ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response , 2000, Nature.

[52]  Y. A. Minamishima,et al.  Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice. , 2000, Genes & development.

[53]  K. Khanna,et al.  Caffeine Abolishes the Mammalian G2/M DNA Damage Checkpoint by Inhibiting Ataxia-Telangiectasia-mutated Kinase Activity* , 2000, The Journal of Biological Chemistry.

[54]  S. Elledge,et al.  Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress. , 2000, Genes & development.

[55]  M. Gatei,et al.  ATM-dependent phosphorylation of nibrin in response to radiation exposure , 2000, Nature Genetics.

[56]  M. Hande,et al.  Characterization of ataxia telangiectasia fibroblasts with extended life-span through telomerase expression , 2001, Oncogene.

[57]  R. Abraham Cell cycle checkpoint signaling through the ATM and ATR kinases. , 2001, Genes & development.

[58]  H. Piwnica-Worms,et al.  ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1 , 2001, Molecular and Cellular Biology.

[59]  R. Paules,et al.  The human decatenation checkpoint , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[60]  S. Schreiber,et al.  ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[61]  N. Mailand,et al.  The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis , 2001, Nature.

[62]  C. Smythe,et al.  Activation of mammalian Chk1 during DNA replication arrest , 2001, The Journal of cell biology.

[63]  E. Appella,et al.  Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase , 2001, Nature.

[64]  W. Kaufmann,et al.  Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant. , 2001, Carcinogenesis.

[65]  R. Paules,et al.  The Ataxia telangiectasia Gene Product Is Required for Oxidative Stress-induced G1 and G2Checkpoint Function in Human Fibroblasts* , 2001, The Journal of Biological Chemistry.

[66]  W. Kaufmann,et al.  DNA damage responses protect xeroderma pigmentosum variant from UVC-induced clastogenesis. , 2002, Carcinogenesis.

[67]  D. Faller,et al.  Carcinogen-induced S-phase arrest is Chk1 mediated and caffeine sensitive. , 2002, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[68]  J. Bartek,et al.  The DNA damage-dependent intra–S phase checkpoint is regulated by parallel pathways , 2002, Nature Genetics.