Phosphoproteomics reveals distinct modes of Mec1/ATR signaling during DNA replication.
The Mec1/Tel1 kinases (human ATR/ATM) play numerous roles in the DNA replication stress response. Despite the multi-functionality of these kinases, studies of their in vivo action have mostly relied on a few well-established substrates. Here we employed a combined genetic-phosphoproteomic approach to monitor Mec1/Tel1 signaling in a systematic, unbiased, and quantitative manner. Unexpectedly, we find that Mec1 is highly active during normal DNA replication, at levels comparable or higher than Mec1's activation state induced by replication stress. This "replication-correlated" mode of Mec1 action requires the 9-1-1 clamp and the Dna2 lagging-strand factor and is distinguishable from Mec1's action in activating the downstream kinase Rad53. We propose that Mec1/ATR performs key functions during ongoing DNA synthesis that are distinct from their canonical checkpoint role during replication stress.
Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Francisco Meirelles
FM
Bastos de Oliveira
Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Dongsung
D
Kim
Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
José Renato
JR
Cussiol
Department of Biological Statistics and Computational Biology, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Jishnu
J
Das
Department of Biological Statistics and Computational Biology, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Min Cheol
MC
Jeong
Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA.
Lillian
L
Doerfler
Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA; Cancer Biology and Evolution Program, H. Lee Moffitt Cancer and Research Institute, Tampa, FL 33612, USA.
Kristina Hildegard
KH
Schmidt
Department of Biological Statistics and Computational Biology, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Haiyuan
H
Yu
Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA. Electronic address: mbs266@cornell.edu.
Marcus Bustamante
MB
Smolka
D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D050783:Proto-Oncogene Proteins c-ets / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000097764:ETS Translocation Variant 6 Protein
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000097764:ETS Translocation Variant 6 Protein; C440013:Ddc1 protein, S cerevisiae; C576597:ATR protein, human; C088937:MEC1 protein, S cerevisiae; C513623:TEL1 protein, S cerevisiae; C067022:RAD53 protein, S cerevisiae