Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Jena, Germany.
PLoS Genet. 2013;9(8):e1003702. doi: 10.1371/journal.pgen.1003702. Epub 2013 Aug 8.
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.
ATR 的激活依赖于与伴侣蛋白的时空相互作用。在出芽酵母模型中，三种蛋白质——Dpb11（TopBP1）、Ddc1（Rad9）和 Dna2——都与并激活 Mec1（ATR）相互作用。每个都包含一个 ATR 激活结构域（ADD），该结构域与 Mec1（ATR）：Ddc2（ATRIP）复合物直接相互作用。任何 Dpb11（TopBP1）、Ddc1（Rad9）或 Dna2 ADD 都足以在体外激活 Mec1（ATR）。这三种都可以独立地在体内激活 Mec1（ATR）：只有当所有三个 AAD 都缺失时，检查点才会丢失。在后生动物中，只有 TopBP1 被鉴定为直接的 ATR 激活剂。耗尽-替换方法表明，TopBP1-AAD 对于 ATR 激活既足够又必要。然而，TopBP1 AAD 的生理功能尚不清楚。我们创建了一个敲入点突变（W1147R），该突变使小鼠 TopBP1-AAD 功能丧失。TopBP1-W1147R 是早期胚胎致死的。为了分析体内 TopBP1-W1147R 的细胞功能，我们在杂合 MEF 中沉默了野生型 TopBP1 等位基因。AAD 失活会损害细胞增殖，促进过早衰老，并在 UV 照射后损害 Chk1 信号传导。我们还表明，强制 TopBP1 二聚化会促进 ATR 依赖性 Chk1 磷酸化。我们的数据表明，与酵母模型不同，TopBP1-AAD 是 ATR 的主要激活剂，维持细胞增殖和胚胎发育。
