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.
DNA damage checkpoint signalling is an essential component of the DNA damage response. Many of the key proteins initiating the checkpoint signal have been identified and characterised in yeast. Here we explore the role of the ATR activating domain (AAD) of TopBP1 in embryonic development, cell growth and checkpoint activation using a mouse model. In contrast to yeasts, where the TopBP1 AAD plays a redundant, and thus phenotypically minor, role in ATR activation, our data demonstrate that the mouse TopBP1 AAD is essential for cellular proliferation. Interestingly, this suggests evolution has provided a simpler ATR activation mechanism in metazoans than it has in yeasts.