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.