Quantitative proteomic analysis of chromatin reveals that Ctf18 acts in the DNA replication checkpoint

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Abstract

Yeast cells lacking Ctf18, the major subunit of an alternative Replication Factor C complex, have multiple problems with genome stability. To understand the in vivo function of the Ctf18 complex, we analyzed chromatin composition in a ctf18Delta mutant using the quantitative proteomic technique of stable isotope labeling by amino acids in cell culture. Three hundred and seven of the 491 reported chromosomal proteins were quantitated. The most marked abnormalities occurred when cells were challenged with the replication inhibitor hydroxyurea. Compared with wild type, hydroxyurea-treated ctf18Delta cells exhibited increased chromatin association of replisome progression complex components including Cdc45, Ctf4, and GINS complex subunits, the polymerase processivity clamp PCNA and the single-stranded DNA-binding complex RPA. Chromatin composition abnormalities observed in ctf18Delta cells were very similar to those of an mrc1Delta mutant, which is defective in the activating the Rad53 checkpoint kinase in response to DNA replication stress. We found that ctf18Delta cells are also defective in Rad53 activation, revealing that the Ctf18 complex is required for engagement of the DNA replication checkpoint. Inappropriate initiation of replication at late origins, because of loss of the checkpoint, probably causes the elevated level of chromatin-bound replisome proteins in the ctf18Delta mutant. The role of Ctf18 in checkpoint activation is not shared by all Replication Factor C-like complexes, because proteomic analysis revealed that cells lacking Elg1 (the major subunit of a different Replication Factor C-like complex) display a different spectrum of chromatin abnormalities. Identification of Ctf18 as a checkpoint protein highlights the usefulness of chromatin proteomic analysis for understanding the in vivo function of proteins that mediate chromatin transactions.
Original languageEnglish
Article numberM110 005561
JournalMolecular and Cellular Proteomics
Volume10
Issue number7
Early online date19 Apr 2011
DOIs
Publication statusPublished - 1 Jul 2011

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DNA Replication
Proteomics
Chromatin
Replication Protein C
DNA
Hydroxyurea
Proteins
Chemical activation
Isotope Labeling
Genomic Instability
Single-Stranded DNA
Clamping devices
Proliferating Cell Nuclear Antigen
Chemical analysis
Cell culture
Isotopes
Yeast
Labeling
Phosphotransferases
Cell Culture Techniques

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Quantitative proteomic analysis of chromatin reveals that Ctf18 acts in the DNA replication checkpoint. / Kubota, Takashi; Hiraga, Shinichiro; Yamada, Kayo; Lamond, A. I.; Donaldson, Anne Dunlop.

In: Molecular and Cellular Proteomics, Vol. 10, No. 7, M110 005561, 01.07.2011.

Research output: Contribution to journalArticle

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