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PNAS 99 (20): 12997-13002

Copyright © 2002 by the National Academy of Sciences.


A dominant-negative MEC3 mutant uncovers new functions for the Rad17 complex and Tel1

Michele Giannattasio*,{dagger}, Elena Sommariva*, Raffaella Vercillo*, Filippo Lippi-Boncambi*, Giordano Liberi*,{dagger}, Marco Foiani*,{dagger}, Paolo Plevani*,{dagger},{ddagger}, and Marco Muzi-Falconi*,{dagger},{ddagger}

*Dipartimento di Genetica e Biologia dei Microrganismi, Universitá degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy; and {dagger}Istituto di Oncologia Molecolare delle Fondazione Italiana Ricerca sul Cancro, Via Adamello 16, 20139 Milan, Italy

Accepted for publication August 2, 2002.

Received for publication February 1, 2002.

Abstract: The Rad17-Mec3-Ddc1 complex is essential for the cellular response to genotoxic agents and is thought to be important for sensing DNA lesions. Deletion of any of the RAD17, MEC3 or DDC1 genes abolishes the G1 and G2 and impairs the intra-S DNA-damage checkpoints. We characterize a dominant-negative mec3-dn mutation that has an unexpected phenotype. It inactivates the G1 checkpoint while it leaves the G2 response functional, thus revealing a difference in the requirements of the DNA-damage response in different phases of the cell cycle. In an attempt to identify the molecular defect imparted by the mutation, we dissected step-by-step the signaling cascade, which is triggered by DNA lesions and requires the activity of Mec1 and Rad53 kinases. The analysis of the phosphorylation state of checkpoint factors and critical protein interactions showed that, in mec3-dn cells, the signal transduction cascade is triggered normally, and the central kinase Mec1 can be activated. In G1 cells expressing the mutation, the signaling cannot proceed any further along the pathway, indicating that the Rad17 complex acts after the activation of Mec1, possibly recruiting targets for the kinase. We also show that the function of the G2 checkpoint in mutant cells is maintained by an uncharacterized activity of Tel1, the yeast homologue of ATM. This work thus reports a previously undiscovered role for Tel1 in checkpoint control.

{ddagger} To whom reprint requests may be addressed. E-mail: marco.muzifalconi{at} or paolo.plevani{at}

Communicated by Thomas J. Kelly, The Sloan–Kettering Institute, New York, NY

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