Ddc2ATRIP promotes Mec1ATR activation at RPA-ssDNA tracts
Ddc2ATRIP promotes Mec1ATR activation at RPA-ssDNA tracts
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DOI：
10.1371/journal.pgen.1008294
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发表时间：
2019-08-01
期刊：
影响因子：
4.5
通讯作者：
Sugimoto, Katsunori
中科院分区：
文献类型：
--
作者：
Biswas, Himadri;Goto, Greicy;Sugimoto, Katsunori
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Author summary The ATR-ATRIP complex is recruited to sites of DNA damage by replication protein A-bound single-stranded DNA (RPA-ssDNA), and controls cellular responses to various types of DNA damage. The recruitment of ATR-ATRIP to RPA-ssDNA is not sufficient for the kinase activation. The activation of ATR-ATRIP requires activators including TopBP1 and ETAA1. In budding yeast, Mec1-Ddc2 (the ATR-ATRIP homolog) is activated through a similar mechanism. Activation of Mec1-Ddc2 requires Ddc1 (Rad9 homolog), Dpb11 (TopBP1 homolog) and Dna2. Our studies of the ddc2-S4 mutation have shown that Ddc2 promotes not only Mec1 recruitment but also Mec1 activation. In this study we show that Ddc2 promotes Mec1 activation independently of Ddc1/Dpb11/Dna2 function in vivo. We also show that Ddc2 regulates Mec1 activation through ssDNA recognition in vitro. Although RPA does not directly stimulate Mec1 activity, it can enhance ssDNA-dependent Mec1 activation. Our results suggest that ATR/Mec1, like ATM/Tel1 and DNA-PKcs, is activated upon DNA damage recognition.The DNA damage checkpoint response is controlled by the phosphatidylinositol 3-kinase-related kinases (PIKK), including ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR). ATR forms a complex with its partner ATRIP. In budding yeast, ATR and ATRIP correspond to Mec1 and Ddc2, respectively. ATRIP/Ddc2 interacts with replication protein A-bound single-stranded DNA (RPA-ssDNA) and recruits ATR/Mec1 to sites of DNA damage. Mec1 is stimulated by the canonical activators including Ddc1, Dpb11 and Dna2. We have characterized the ddc2-S4 mutation and shown that Ddc2 not only recruits Mec1 to sites of DNA damage but also stimulates Mec1 kinase activity. However, the underlying mechanism of Ddc2-dependent Mec1 activation remains to be elucidated. Here we show that Ddc2 promotes Mec1 activation independently of Ddc1/Dpb11/Dna2 function in vivo and through ssDNA recognition in vitro. The ddc2-S4 mutation diminishes damage-induced phosphorylation of the checkpoint mediators, Rad9 and Mrc1. Rad9 controls checkpoint throughout the cell-cycle whereas Mrc1 is specifically required for the S-phase checkpoint. Notably, S-phase checkpoint signaling is more defective in ddc2-S4 mutants than in cells where the Mec1 activators (Ddc1/Dpb11 and Dna2) are dysfunctional. To understand a role of Ddc2 in Mec1 activation, we reconstituted an in vitro assay using purified Mec1-Ddc2 complex, RPA and ssDNA. Whereas ssDNA stimulates kinase activity of the Mec1-Ddc2 complex, RPA does not. However, RPA can promote ssDNA-dependent Mec1 activation. Neither ssDNA nor RPA-ssDNA efficiently stimulates the Mec1-Ddc2 complex containing Ddc2-S4 mutant. Together, our data support a model in which Ddc2 promotes Mec1 activation at RPA-ssDNA tracts.