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 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.
Highlights
Cells deploy multiple interconnected pathways to ensure accurate chromosome maintenance, especially during DNA replication when the unwound DNA helix is vulnerable to DNA damage
Workflow for quantitative analysis of chromatin components in wild-type and ctf18! cells! To understand the in vivo function of Ctf18-RFC-like complex (RLC), we used SILAC-based comparative proteomics [30] to compare chromatin composition in wild-type and ctf18! cells (Fig. 1A). ctf18! cells were grown in 'heavy' media, i.e., containing 13C/15N-substituted arginine and/or lysine; complete labeling of cellular proteins was facilitated by the use of lysine and arginine auxotrophic mutants
Chromatin proteins were prepared from wild-type and ctf18! cells separately, mixed and size-fractionated using SDS-PAGE, followed by trypsin or Lys-C digestion
Summary
Cells deploy multiple interconnected pathways to ensure accurate chromosome maintenance, especially during DNA replication when the unwound DNA helix is vulnerable to DNA damage. PCNA is central to the replication machinery and is a multifunctional complex, acting as a platform that interacts with many proteins including DNA polymerases, DNA helicases, nucleases, DNA ligases, histone chaperones, DNA repair proteins and sister chromatid cohesion factors [3]. All eukaryotic cells have a series of RFC-like complexes These 'RLC' complexes share the Rfc subunits with RFC, but Rfc is replaced by one of a series of 'alternative' subunits: Rad (called Rad in human), Elg, or Ctf18 [4]. Rad24-RLC is the best understood, and acts to load the PCNA-like complex Rad17-Mec3-Ddc (the equivalent of the human 9-1-1 complex) at DNA damage sites. Elg1-RLC binds PCNA, but has not been reported to load or unload it on DNA. Ctf18-RLC appears to act at DNA replication forks [16, 17], the in vivo function of Ctf18-RLC is unknown
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