2012年7月5日,，北京生命科學(xué)研究所杜立林實(shí)驗(yàn)室在《PLoS Genetics》在線發(fā)表題為“Phosphorylation-Dependent Interactions between Crb2 and Chk1 Are Essential for DNA Damage Checkpoint”的文章,。
為確保基因組的穩(wěn)定性,，真核細(xì)胞中存在一套監(jiān)控DNA完整性并傳遞DNA損傷信號(hào)的系統(tǒng),，稱為 DNA損傷檢驗(yàn)點(diǎn)通路。參與該信號(hào)傳導(dǎo)通路的蛋白包括識(shí)別DNA損傷的感應(yīng)蛋白,，下游的效應(yīng)激酶,，還有介導(dǎo)感應(yīng)蛋白與效應(yīng)激酶間信號(hào)傳導(dǎo)的中介蛋白。Chk1是最重要的效應(yīng)激酶之一,，在真核生物中高度保守,。在模式生物裂殖酵母中,，中介蛋白Crb2對(duì)效應(yīng)激酶Chk1的激活至關(guān)重要，但是Crb2參與激活Chk1的分子機(jī)理卻并不清楚,。本論文對(duì)這一問題進(jìn)行了研究,，發(fā)現(xiàn)Crb2上的兩個(gè)磷酸化位點(diǎn)（T73和S80）對(duì)于Chk1在DNA損傷部位的聚集和Chk1的激活是必需的。一個(gè)包含這兩個(gè)磷酸化位點(diǎn)的19個(gè)氨基酸的肽段可以在體外與Chk1直接結(jié)合,。通過將這一肽段定位在DNA損傷部位,，本論文證明了Crb2和另一個(gè)中介蛋白復(fù)合體Rad9-Rad1-Hus1 (9-1-1)的主要作用是將Chk1募集到DNA損傷部位。
本研究工作主要由我所博士生曲萌完成,。其他作者包括我所的董夢(mèng)秋博士,，董夢(mèng)秋實(shí)驗(yàn)室的博士生楊兵和陶莉，以及美國Scripps研究所的Paul Russell博士和John R. Yates III博士,。杜立林博士為本文的通訊作者,。此項(xiàng)研究由科技部和北京市政府資助。（生物谷Bioon.com）
doi:10.1371/journal.pgen.1002817
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Phosphorylation-Dependent Interactions between Crb2 and Chk1 Are Essential for DNA Damage Checkpoint
Meng Qu1,2, Bing Yang2, Li Tao2, John R. Yates III3, Paul Russell4, Meng-Qiu Dong2, Li-Lin Du2*
In response to DNA damage, the eukaryotic genome surveillance system activates a checkpoint kinase cascade. In the fission yeast Schizosaccharomyces pombe, checkpoint protein Crb2 is essential for DNA damage-induced activation of downstream effector kinase Chk1. The mechanism by which Crb2 mediates Chk1 activation is unknown. Here, we show that Crb2 recruits Chk1 to double-strand breaks (DSBs) through a direct physical interaction. A pair of conserved SQ/TQ motifs in Crb2, which are consensus phosphorylation sites of upstream kinase Rad3, is required for Chk1 recruitment and activation. Mutating both of these motifs renders Crb2 defective in activating Chk1. Tethering Crb2 and Chk1 together can rescue the SQ/TQ mutations, suggesting that the main function of these phosphorylation sites is promoting interactions between Crb2 and Chk1. A 19-amino-acid peptide containing these SQ/TQ motifs is sufficient for Chk1 binding in vitro when one of the motifs is phosphorylated. Remarkably, the same peptide, when tethered to DSBs by fusing with either recombination protein Rad22/Rad52 or multi-functional scaffolding protein Rad4/Cut5, can rescue the checkpoint defect of crb2Δ. The Rad22 fusion can even bypass the need for Rad9-Rad1-Hus1 (9-1-1) complex in checkpoint activation. These results suggest that the main role of Crb2 and 9-1-1 in DNA damage checkpoint signaling is recruiting Chk1 to sites of DNA lesions.
