title: Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus egg extracts requires binding of ATRIP to ATR but not the stable DNA-binding or coiled-coil domains of ATRIP.
abstract: ATR, a critical regulator of DNA replication and damage checkpoint responses, possesses a binding partner called ATRIP. We have studied the functional properties of Xenopus ATR and ATRIP in incubations with purified components and in frog egg extracts. In purified systems, ATRIP associates with DNA in both RPA-dependent and RPA-independent manners, depending on the composition of the template. However, in egg extracts, only the RPA-dependent mode of binding to DNA can be detected. ATRIP adopts an oligomeric state in egg extracts that depends upon binding to ATR. In addition, ATR and ATRIP are mutually dependent on one another for stable binding to DNA in egg extracts. The ATR-dependent oligomerization of ATRIP does not require an intact coiled-coil domain in ATRIP and does not change in the presence of checkpoint-inducing DNA templates. Egg extracts containing a mutant of ATRIP that cannot bind to ATR are defective in the phosphorylation of Chk1. However, extracts containing mutants of ATRIP lacking stable DNA-binding and coiled-coil domains show no reduction in the phosphorylation of Chk1 in response to defined DNA templates. Furthermore, activation of Chk1 does not depend upon RPA under these conditions. These results suggest that ATRIP must associate with ATR in order for ATR to carry out the phosphorylation of Chk1 effectively. However, this function of ATRIP does not involve its ability to mediate the stable binding of ATR to defined checkpoint-inducing DNA templates in egg extracts, does not require an intact coiled-coil domain, and does not depend on RPA.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Soo-Mi
authors.initials: SM
authors.lastname: Kim
authors.forename: Akiko
authors.initials: A
authors.lastname: Kumagai
authors.forename: Joon
authors.initials: J
authors.lastname: Lee
authors.forename: William G
authors.initials: WG
authors.lastname: Dunphy
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D000071877:Checkpoint Kinase 1; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry; D007313:Insecta; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*
chemical_list: C495984:ATRIP protein, Xenopus; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D004247:DNA; D019809:Streptavidin; D011494:Protein Kinases; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases