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.2001 Sep;21(17):5838-45.
doi: 10.1128/MCB.21.17.5838-5845.2001.

Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway

Affiliations

Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway

T Naiki et al. Mol Cell Biol.2001 Sep.

Abstract

RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24Delta mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12Delta nor rad24Delta single mutants are defective, chl12Delta rad24Delta double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.

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Figures

FIG. 1
FIG. 1
Structures of theS. cerevisiae Chl12, Rad24, and RFC proteins. There are eight RFC boxes numbered consecutively from the amino terminus to the carboxyl terminus. All of the RFC proteins possess the RFC boxes II to VIII. Box I is present only in the largest RFC subunits. The solid and shaded boxes indicate high and moderate degrees of homology, respectively. a.a., amino acids.
FIG. 2
FIG. 2
Viability ofchl12Δ andchl12Δ rad24Δ mutants following exposure to HU, MMS, or UV light. Wild-type (KSC006),chl12Δ (KSC1148),rad24Δ (KSC1090), andchl12Δ rad24Δ (KSC1266) cells were grown in log phase at 30°C, treated with HU or MMS, and irradiated with UV light. The viability of the cells was estimated as described in Materials and Methods.
FIG. 3
FIG. 3
DNA replication block checkpoint inchl12Δ rad24Δ mutants. Cells were arrested at G1 with α-factor and then released in YEPD with or without 10 mg of HU/ml at 30°C. Aliquots of cells were collected at the indicated times and stained with anti-tubulin antibodies. The percentage of cells with elongated spindles was scored as described in Materials and Methods. The strains used were the wild type (KSC006),chl12Δ (KSC1148),rad24Δ (KSC1090), andchl12Δ rad24Δ (KSC1266).
FIG. 4
FIG. 4
Effect ofchl12Δ rad24Δ mutation on Rad53 modification following replication block. Cells carrying YCpRAD53-HA were grown at 30°C, arrested in G1 with α-factor, and then released in YEPD containing 5 mg of HU/ml. Aliquots of cells were collected at the indicated times and subjected to immunoblotting analysis as described in Materials and Methods. The strains used were the wild-type (KSC006),chl12Δ (KSC1148),rad24Δ (KSC1090), andchl12Δ rad24Δ (KSC1266).
FIG. 5
FIG. 5
DNA damage checkpoints inchl12Δ andchl12Δ rad24Δ mutants. (A) G2/M-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were grown at 30°C, arrested with nocodazole, and treated or not treated with MMS. At the indicated times after release of MMS-treated (+MMS) and untreated (−MMS) cultures from nocodazole, the percentage of uninucleate large budded cells was scored by DAPI staining. (B) S-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were synchronized with α-factor in G1 and released in either the presence or the absence of MMS at 30°C as described in Materials and Methods. Aliquots of cells were collected at the indicated times after release from α-factor treatment and examined for DNA content by flow cytometry. The dotted lines indicate the DNA content of 1C and 2C cells. The top panels represent asynchronous (As) cells not treated with MMS at 30°C and are included as a reference. (C) G1-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were synchronized with α-factor in G1 and treated with MMS (+MMS) or not treated (−MMS). At the indicated times after release from α-factor, the percentage of small budded cells was scored under microscopy. The strains used are the wild type (KSC006),chl12Δ (KSC1148),rad24Δ (KSC1090), andchl12Δ rad24Δ (KSC1266).
FIG. 5
FIG. 5
DNA damage checkpoints inchl12Δ andchl12Δ rad24Δ mutants. (A) G2/M-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were grown at 30°C, arrested with nocodazole, and treated or not treated with MMS. At the indicated times after release of MMS-treated (+MMS) and untreated (−MMS) cultures from nocodazole, the percentage of uninucleate large budded cells was scored by DAPI staining. (B) S-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were synchronized with α-factor in G1 and released in either the presence or the absence of MMS at 30°C as described in Materials and Methods. Aliquots of cells were collected at the indicated times after release from α-factor treatment and examined for DNA content by flow cytometry. The dotted lines indicate the DNA content of 1C and 2C cells. The top panels represent asynchronous (As) cells not treated with MMS at 30°C and are included as a reference. (C) G1-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were synchronized with α-factor in G1 and treated with MMS (+MMS) or not treated (−MMS). At the indicated times after release from α-factor, the percentage of small budded cells was scored under microscopy. The strains used are the wild type (KSC006),chl12Δ (KSC1148),rad24Δ (KSC1090), andchl12Δ rad24Δ (KSC1266).
FIG. 5
FIG. 5
DNA damage checkpoints inchl12Δ andchl12Δ rad24Δ mutants. (A) G2/M-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were grown at 30°C, arrested with nocodazole, and treated or not treated with MMS. At the indicated times after release of MMS-treated (+MMS) and untreated (−MMS) cultures from nocodazole, the percentage of uninucleate large budded cells was scored by DAPI staining. (B) S-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were synchronized with α-factor in G1 and released in either the presence or the absence of MMS at 30°C as described in Materials and Methods. Aliquots of cells were collected at the indicated times after release from α-factor treatment and examined for DNA content by flow cytometry. The dotted lines indicate the DNA content of 1C and 2C cells. The top panels represent asynchronous (As) cells not treated with MMS at 30°C and are included as a reference. (C) G1-phase DNA damage checkpoint inchl12Δ andchl12Δ rad24Δ mutants. Cells were synchronized with α-factor in G1 and treated with MMS (+MMS) or not treated (−MMS). At the indicated times after release from α-factor, the percentage of small budded cells was scored under microscopy. The strains used are the wild type (KSC006),chl12Δ (KSC1148),rad24Δ (KSC1090), andchl12Δ rad24Δ (KSC1266).
FIG. 6
FIG. 6
Physical interaction of Chl12 with Rfc2, Rfc3, Rfc4, and Rfc5. (A and B) Interaction of Chl12 with Rfc2, Rfc3, Rfc4, and Rfc5. Extracts were prepared fromCHL12-HA cells containing no FLAG construct or the indicated FLAG-tagged construct (A) or cells containing the indicated FLAG-tagged construct and no HA construct (−) orCHL12-HA (+) (B) and subjected to immunoprecipitation (IP) with anti-FLAG antibody (A) or anti-HA antibody (B). An F after a gene name indicates the addition of FLAG epitopes. The immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-FLAG or anti-HA antibody. Whole extracts were immunoblotted with anti-HA antibody (A) or anti-FLAG (B) antibody. (C) Interaction of Chl12 with Rad24 and Rfc1. Extracts were prepared fromRAD24-FLAG andRFC1-FLAG cells containing no HA construct (−) orCHL12-HA (+) and subjected to IP and immunoblotting analysis as for panel A. (D) Interaction of Chl12 with Rfc3 inRFC5 andrfc5-1 mutant cells. Extracts were prepared fromRFC5 orrfc5-1 cells containingCHL12-HA andRFC3-FLAG and were subjected to IP and immunoblotting analysis as for panel B.
FIG. 7
FIG. 7
Sedimentation of Chl12, Rad24, and Rfc1 in sucrose density gradient centrifugation. Extracts were prepared fromCHL12-HA RAD24-myc RFC1-FLAG (KSC1433) cells and separated by centrifugation in a 10 to 40% sucrose gradient for 16 h. The load on the gradient (L) and fractions (removed from the top of the gradient) were analyzed by immunoblotting using anti-FLAG, anti-HA, or anti-myc antibody. An F after a gene name indicates the addition of FLAG epitopes. Bovine serum albumin (4.5S) and thyroglobulin (16.5–19S) were separated simultaneously in an independent gradient as markers.
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