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 ctf18Δ 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 ctf18Δ 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 ctf18Δ cells were very similar to those of an mrc1Δ mutant, which is defective in the activating the Rad53 checkpoint
kinase in response to DNA replication stress. We found that ctf18Δ 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 ctf18Δ 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.