ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation.

ATR associates with the regulatory protein ATRIP that has been proposed to localize ATR to sites of DNA damage through an interaction with single-stranded DNA (ssDNA) coated with replication protein A (RPA). We tested this hypothesis and found that ATRIP is required for ATR accumulation at intranuclear foci induced by DNA damage. A domain at the N terminus of ATRIP is necessary and sufficient for interaction with RPA-ssDNA. Deletion of the ssDNA-RPA interaction domain of ATRIP greatly diminished accumulation of ATRIP into foci. However, the ATRIP-RPA-ssDNA interaction is not sufficient for ATRIP recognition of DNA damage. A splice variant of ATRIP that cannot bind to ATR revealed that ATR association is also essential for proper ATRIP localization. Furthermore, the ATRIP-RPA-ssDNA interaction is not absolutely essential for ATR activation because ATR phosphorylates Chk1 in cells expressing only a mutant of ATRIP that does not bind to RPA-ssDNA. These data suggest that binding to RPA-ssDNA is not the essential function of ATRIP in ATR-dependent checkpoint signaling and ATR has an important function in properly localizing the ATR-ATRIP complex.

[1]  Soo-Mi Kim,et al.  Claspin and the Activated Form of ATR-ATRIP Collaborate in the Activation of Chk1* , 2004, Journal of Biological Chemistry.

[2]  R. Abraham,et al.  Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling , 2004, The EMBO journal.

[3]  M. Kimura,et al.  Amino‐terminal domain of ATRIP contributes to intranuclear relocation of the ATR–ATRIP complex following DNA damage , 2004, FEBS letters.

[4]  R. Rothstein,et al.  Choreography of the DNA Damage Response Spatiotemporal Relationships among Checkpoint and Repair Proteins , 2004, Cell.

[5]  R. Tibbetts,et al.  DNA Replication Defects, Spontaneous DNA Damage, and ATM-dependent Checkpoint Activation in Replication Protein A-deficient Cells* , 2004, Journal of Biological Chemistry.

[6]  S. Elledge,et al.  Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Gautier,et al.  ATR and ATM regulate the timing of DNA replication origin firing , 2004, Nature Cell Biology.

[8]  W. Chazin,et al.  Physical Interaction between Replication Protein A and Rad51 Promotes Exchange on Single-stranded DNA* , 2004, Journal of Biological Chemistry.

[9]  N. Lakin,et al.  Recruitment of the Cell Cycle Checkpoint Kinase ATR to Chromatin during S-phase* , 2004, Journal of Biological Chemistry.

[10]  K. Cimprich,et al.  A Novel Protein Activity Mediates DNA Binding of an ATR-ATRIP Complex* , 2004, Journal of Biological Chemistry.

[11]  A. Sancar,et al.  Quaternary Structure of ATR and Effects of ATRIP and Replication Protein A on Its DNA Binding and Kinase Activities , 2004, Molecular and Cellular Biology.

[12]  L. Karnitz,et al.  Cut5 Is Required for the Binding of Atr and DNA Polymerase α to Genotoxin-damaged Chromatin* , 2003, Journal of Biological Chemistry.

[13]  S. Elledge,et al.  Replication protein A-mediated recruitment and activation of Rad17 complexes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  K. Cimprich,et al.  ATR Kinase Activity Regulates the Intranuclear Translocation of ATR and RPA following Ionizing Radiation , 2003, Current Biology.

[15]  Stephen J. Elledge,et al.  Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes , 2003, Science.

[16]  J. Diffley,et al.  A central role for DNA replication forks in checkpoint activation and response. , 2003, Molecular cell.

[17]  A. Kumagai,et al.  Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. , 2003, Molecular cell.

[18]  J. Gautier,et al.  Single-Strand DNA Gaps Trigger an ATR- and Cdc7-Dependent Checkpoint , 2003, Cell cycle.

[19]  L. Karnitz,et al.  Cut5 is required for the binding of Atr and DNA polymerase alpha to genotoxin-damaged chromatin. , 2003, The Journal of biological chemistry.

[20]  J. Gautier,et al.  An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication. , 2003, Molecular cell.

[21]  T. Enoch,et al.  Fission yeast Rad26 responds to DNA damage independently of Rad3 , 2003, BMC Genetics.

[22]  K. Cimprich,et al.  A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. , 2002, Genes & development.

[23]  J. Newport,et al.  The Role of Single-stranded DNA and Polymerase α in Establishing the ATR, Hus1 DNA Replication Checkpoint* , 2002, The Journal of Biological Chemistry.

[24]  J. Griffith,et al.  Preferential binding of ATR protein to UV-damaged DNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[25]  John Rouse,et al.  Lcd1p recruits Mec1p to DNA lesions in vitro and in vivo. , 2002, Molecular cell.

[26]  Jun Qin,et al.  ATR and ATRIP: Partners in Checkpoint Signaling , 2001, Science.

[27]  D. Toczyski,et al.  Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. , 2001, Genes & development.

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

[29]  Katsunori Sugimoto,et al.  Pie1, a Protein Interacting with Mec1, Controls Cell Growth and Checkpoint Responses in Saccharomyces cerevisiae , 2001, Molecular and Cellular Biology.

[30]  Y. Shiloh.  ATM and ATR: networking cellular responses to DNA damage. , 2001, Current opinion in genetics & development.

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

[32]  S. Jackson,et al.  LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae , 2000, The EMBO journal.

[33]  E. Fanning,et al.  Activation of the DNA replication checkpoint through RNA synthesis by primase. , 2000, Science.

[34]  G. Lucchini,et al.  The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast. , 2000, Genes & development.

[35]  S. Elledge,et al.  BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. , 2000, Genes & development.

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

[37]  A. Carr,et al.  A Rad3–Rad26 complex responds to DNA damage independently of other checkpoint proteins , 1999, Nature Cell Biology.

[38]  I. Galli,et al.  A novel mutant allele of Schizosaccharomyces pombe rad26 defective in monitoring S-phase progression to prevent premature mitosis , 1997, Molecular and cellular biology.

[39]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.