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J. Biol. Chem., Vol. 278, Issue 25, 22303-22308, June 20, 2003

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Correlation between Checkpoint Activation and in Vivo Assembly of the Yeast Checkpoint Complex Rad17-Mec3-Ddc1^*

Michele Giannattasio, Simone Sabbioneda, Mario Minuzzo, Paolo Plevani  and Marco Muzi-Falconi 

From the Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy

Rad17-Mec3-Ddc1 forms a proliferating cell nuclear antigen-like complex that is required for the DNA damage response in Saccharomyces cerevisiae and acts at an early step of the signal transduction cascade activated by DNA lesions. We used the mec3-dn allele, which causes a dominant negative checkpoint defect in G₁ but not in G₂, to test the stability of the complex in vivo and to correlate its assembly and disassembly with the mechanisms controlling checkpoint activation. Under physiological conditions, the mutant complex is formed both in G₁ and G₂, although the mutant phenotype is detectable only in G₁, suggesting that is not the presence of the mutant complex per se to cause a checkpoint defect. Our data indicate that the Rad17-Mec3-Ddc1 complex is very stable, and it takes several hours to replace Mec3 with Mec3-dn within a wild type complex. On the other hand, the mutant complex is rapidly assembled when starting from a condition where the complex is not pre-assembled, indicating that the critical factor for the substitution is the disassembly step rather than complex formation. Moreover, the kinetics of mutant complex assembly, starting from conditions in which the wild type form is present, parallels the kinetics of checkpoint inactivation, suggesting that the complex acts in a stoichiometric way, rather than catalytically.

Received for publication, February 5, 2003 , and in revised form, March 24, 2003.

^* This work was supported by grants from AIRC, MIUR (5%) Biomolecole per la Salute Umana, MIUR "Genomica Funzionale," and Progetto FIRB-MIUR "Genomica e Proteomica Nello Studio di Funzioni Cellulari Complesse." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy. Tel.: 39-02-50315032; Fax: 39-02-50315044; E-mail: paolo.plevani{at}unimi.it.

To whom correspondence may be addressed: Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy. Tel.: 39-02-50315032; Fax: 39-02-50315044; E-mail: marco.muzifalconi{at}unimi.it.

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