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.

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Journal Article | Research Support, N.I.H., Extramural

Authors

Biswas H, Goto G, Wang W, Sung P, Sugimoto K

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