An essential function for the ATR-activation-domain (AAD) of TopBP1 in mouse development and cellular senescence.
ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins - Dpb11(TopBP1), Ddc1(Rad9) and Dna2 - all interact with and activate Mec1(ATR). Each contains an ATR activation domain (ADD) that interacts directly with the Mec1(ATR):Ddc2(ATRIP) complex. Any of the Dpb11(TopBP1), Ddc1(Rad9) or Dna2 ADDs is sufficient to activate Mec1(ATR) in vitro. All three can also independently activate Mec1(ATR) in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development.
Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena, Germany.
Zhong-Wei
ZW
Zhou
Cong
C
Liu
Tang-Liang
TL
Li
Christopher
C
Bruhn
Anja
A
Krueger
WooKee
W
Min
Zhao-Qi
ZQ
Wang
Antony M
AM
Carr
D000483:Alleles; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000235:genetics*; D000071877:Checkpoint Kinase 1; D047108:Embryonic Development / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D055879:Gene Knock-In Techniques; D051379:Mice; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction
D002352:Carrier Proteins; C492846:Topbp1 protein, mouse; D011494:Protein Kinases; C576897:Atr protein, mouse; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse