Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts
Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts
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DOI：
10.1101/gad.842500
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发表时间：
2000-11-01
影响因子：
10.5
通讯作者：
Dunphy, WG
中科院分区：
文献类型：
--
作者：
Guo, ZJ;Kumagai, A;Dunphy, WG
检查点激酶Xchk 1在非洲爪蟾卵提取物中响应于DNA复制阻断或UV损伤的DNA而磷酸化。Xchk 1也是由未复制或UV损伤的DNA诱导的细胞周期延迟所必需的。在这份报告中，我们已经删除了非洲爪蟾同系物ATR（Xatr）从鸡蛋提取物的免疫耗竭。在去除Xatr的提取物中，Xchk 1的检查点相关磷酸化被消除，并且由复制阻断诱导的细胞周期延迟被强烈损害。卵提取物中的Xatr在体外磷酸化重组Xchk 1，但不是在其四个保守的SQ/TQ基序中含有不可磷酸化残基的突变形式的Xchk 1（Xchk 1 -4AQ）。重组人ATR，但不是激酶失活突变体，磷酸化Xchk 1中的相同位点。此外，发现Xchk 1 -4AQ突变体在介导蛋提取物中的检查点应答方面存在缺陷。这些发现表明，Xchk 1是Xatr在对未复制或UV损伤的DNA的检查点响应期间的功能重要靶标。
The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts in response to DNA replication blocks or UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. In this report, we have removed the Xenopus homolog of ATR (Xatr) from egg extracts by immunodepletion. In Xatr-depleted extracts, the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is strongly compromised. Xatr from egg extracts phosphorylated recombinant Xchk1 in vitro, but not a mutant form of Xchk1 (Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant, phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts. These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.