Tipin-replication protein A interaction mediates Chk1 phosphorylation by ATR in response to genotoxic stress.
Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Michael G
MG
Kemp
Zafer
Z
Akan
Seçil
S
Yilmaz
Mary
M
Grillo
Stephanie L
SL
Smith-Roe
Tae-Hong
TH
Kang
Marila
M
Cordeiro-Stone
William K
WK
Kaufmann
Robert T
RT
Abraham
Aziz
A
Sancar
Keziban
K
Unsal-Kaçmaz
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins; D019281:Dimerization; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051716:Replication Protein A / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051716:Replication Protein A; C516227:Tipin protein, human; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases