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.

Correlation between checkpoint activation and in vivo assembly of the yeast checkpoint complex Rad17-Mec3-Ddc1 / M. Giannattasio, S. Sabbioneda, M. Minuzzo, P. Plevani, M. Muzi-Falconi. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 278:25(2003 Jun 20), pp. 22303-22308.

Correlation between checkpoint activation and in vivo assembly of the yeast checkpoint complex Rad17-Mec3-Ddc1

M. Giannattasio
Primo
;
S. Sabbioneda
Secondo
;
M. Minuzzo;P. Plevani
Penultimo
;
M. Muzi-Falconi
2003-06-20

Abstract

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.
Cell Cycle Proteins ; DNA-Binding Proteins ; Saccharomyces cerevisiae Proteins ; Saccharomyces cerevisiae ; Phenotype ; Macromolecular Substances ; Nuclear Proteins ; Phosphoproteins ; Kinetics ; Nocodazole ; Cell Cycle ; Time Factors ; Signal Transduction
Settore BIO/11 - Biologia Molecolare
Settore BIO/18 - Genetica
Settore BIO/10 - Biochimica
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/191040
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