title: Two distinct modes of ATR activation orchestrated by Rad17 and Nbs1.
abstract: The ATM- and Rad3-related (ATR) kinase is a master regulator of the DNA damage response, yet how ATR is activated toward different substrates is still poorly understood. Here, we show that ATR phosphorylates Chk1 and RPA32 through distinct mechanisms at replication-associated DNA double-stranded breaks (DSBs). In contrast to the rapid phosphorylation of Chk1, RPA32 is progressively phosphorylated by ATR at Ser33 during DSB resection prior to the phosphorylation of Ser4/Ser8 by DNA-PKcs. Surprisingly, despite its reliance on ATR and TopBP1, substantial RPA32 Ser33 phosphorylation occurs in a Rad17-independent but Nbs1-dependent manner in vivo and in vitro. Importantly, the role of Nbs1 in RPA32 phosphorylation can be separated from ATM activation and DSB resection, and it is dependent upon the interaction of Nbs1 with RPA. An Nbs1 mutant that is unable to bind RPA fails to support proper recovery of collapsed replication forks, suggesting that the Nbs1-mediated mode of ATR activation is important for the repair of replication-associated DSBs.
authors.affiliation: Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA. bshiotan@hiroshima-u.ac.jp
authors.forename: Bunsyo
authors.initials: B
authors.lastname: Shiotani
authors.forename: Hai Dang
authors.initials: HD
authors.lastname: Nguyen
authors.forename: Pelle
authors.initials: P
authors.lastname: Håkansson
authors.forename: Alexandre
authors.initials: A
authors.lastname: Maréchal
authors.forename: Alice
authors.initials: A
authors.lastname: Tse
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Tahara
authors.forename: Lee
authors.initials: L
authors.lastname: Zou
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; C113575:Rad17 protein, human; D051716:Replication Protein A; C110513:TOPBP1 protein, human; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C516794:RPA2 protein, human; D004706:Endodeoxyribonucleases; C410741:RBBP8 protein, human