A Tel1/MRX-Dependent Checkpoint Inhibits the Metaphase-to-Anaphase Transition after UV Irradiation in the Absence of Mec1

Abstract

In Saccharomyces cerevisiae, Mec1/ATR plays a primary role in sensing and transducing checkpoint signals in response to different types of DNA lesions, while the role of the Tel1/ATM kinase in DNA damage checkpoints is not as well defined. We found that UV irradiation in G₁ in the absence of Mec1 activates a Tel1/MRX-dependent checkpoint, which specifically inhibits the metaphase-to-anaphase transition. Activation of this checkpoint leads to phosphorylation of the downstream checkpoint kinases Rad53 and Chk1, which are required for Tel1-dependent cell cycle arrest, and their adaptor Rad9. The spindle assembly checkpoint protein Mad2 also partially contributes to the G₂/M arrest of UV-irradiated mec1Δ cells independently of Rad53 phosphorylation and activation. The inability of UV-irradiated mec1Δ cells to undergo anaphase can be relieved by eliminating the anaphase inhibitor Pds1, whose phosphorylation and stabilization in these cells depend on Tel1, suggesting that Pds1 persistence may be responsible for the inability to undergo anaphase. Moreover, while UV irradiation can trigger Mec1-dependent Rad53 phosphorylation and activation in G₁- and G₂-arrested cells, Tel1-dependent checkpoint activation requires entry into S phase independently of the cell cycle phase at which cells are UV irradiated, and it is decreased when single-stranded DNA signaling is affected by the rfa1-t11 allele. This indicates that UV-damaged DNA molecules need to undergo structural changes in order to activate the Tel1-dependent checkpoint. Active Clb-cyclin-dependent kinase 1 (CDK1) complexes also participate in triggering this checkpoint and are required to maintain both Mec1- and Tel1-dependent Rad53 phosphorylation, suggesting that they may provide critical phosphorylation events in the DNA damage checkpoint cascade.

We thank J. F. X. Diffley, R. D. Kolodner, N. Lowndes, K. Nasmyth, S. Piatti, and F. Uhlmann for providing reagents; S. Piatti for helpful suggestions and critically reading the manuscript; and M. Foiani and all the members of our laboratory for useful discussions and criticisms.

This work was supported in part by grants from Telethon-Italy (E.1247) and the Associazione Italiana per la Ricerca sul Cancro and Cofinanziamento 2003 MIUR/Università di Milano-Bicocca to M.P.L. and grants from the Fondo per gli Investimenti della Ricerca di Base (FIRB) and Progetto Strategico MIUR-Legge 449/97 to G.L. V.B. was supported by a fellowship from Fondazione Italiana per la Ricerca sul Cancro.