1. Identification of RFC(Ctf18p, Ctf8p, Dcc1p)
Melanie L. Mayer, Steven P. Gygi, Ruedi Aebersold, Philip Hieter 
Molecular Cell 
Volume 7, Issue 5, Pages (May 2001)
DOI: /S (01)

2. Figure 1
Ctf8p Is an Evolutionarily Conserved Protein that Associates with Chromatin
(A) Multiple protein sequence alignments of Ctf8p homologs from S. cerevisiae (Sc), C. elegans (Ce), A. thaliana (At), and H. sapiens (Hs). The C. elegans Ctf8p sequence corresponds to GenBank accession number CAA (Worm PD ID T22C1.4). The A. thaliana sequence represents GenBank accession number BAA The human Ctf8p homolog sequence is derived from the translation of a human EST with GenBank accession number AI The location and nature of the three original ctf8 mutations (ctf8-9, ctf8-133, and ctf8-162) in the ctf collection are shown. The human Ctf8p homolog has a putative leucine zipper motif denoted by asterisks.
(B) Two independent wild-type (YPH1466 and YPH1467) and two independent ctf8Δ (YPH1464 and YPH 1465) strains were plated onto plates containing 0 or 15 μg/ml benomyl. Plates were grown at 30°C for 3 days.
(C) Wild-type (YPH1466 and YPH1467) and ctf8Δ (YPH1464 and YPH1465) strains were arrested in G2/M or G1 and then plated onto YPD plates. Viability was assessed based upon the ability of these cells to form microcolonies. The data shown represents the average of two independent experiments. Two hundred cells were counted for each sample.
(D) Homozygous wild-type (YPH1468), ctf8Δ/ctf8Δ (YPH1469), rad9Δ/rad9Δ (YPH1471), mad2Δ/mad2Δ (YPH1470), ctf8Δ/ctf8Δ rad9Δ/rad9Δ (YPH1473), and ctf8Δ/ctf8Δ mad2Δ/mad2Δ (YPH1472) strains were grown to early log phase. DNA content was measured by FACS.
(E and F) Diploid cells expressing either Ctf8p (YPH1468) or Ctf8-13myc (YPH1501) were processed for immunofluorescence or chromosome spreads. DNA was stained with DAPI, and Ctf8-13myc was detected using a monoclonal antibody against the myc epitope (9E10; Boehringer Mannheim) and goat anti-mouse Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories)
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 Ctf8p Is Required for Sister Chromatid Cohesion
(A) The number of GFP signals was scored in wild-type (YPH1477) and two independent ctf8Δ strains (YPH1478 and YPH1479). The data shown represents the average of two independent experiments. One hundred cells were counted for each sample.
(B) The number of GFP signals was scored in wild-type (Y819) and two independent ctf8Δ strains (YPH1497 and YPH1498). The data shown represents the average of two independent experiments. One hundred cells were counted for each sample.
(C) Wild-type (YPH1477) and ctf8Δ (YPH1478 and YPH1479) strains were arrested in G1 with α factor for 3 hr at 25°C and released into YPD containing nocodazole at 25°C. Samples were taken every 20 min for 160 min. DNA content was determined by FACS and the number of GFP signals was scored. The data shown represents the data collected from one experiment and is representative of the data obtained when the experiment was repeated
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Ctf8p Physically Associates with Rfc2p, Rfc3p, Rfc4p, Rfc5p, Ctf18p, and Dcc1p
(A) Anti-myc immunoprecipitations were performed with extract from logarithmically growing (OD(600) = 0.7) 1 L cultures of either Ctf8p (YPH1466), Ctf8-13myc (YPH1474), or Dcc1-13myc (YPH1482) strains. The immunoprecipitated proteins were separated on a 10% SDS-PAGE gel that was subsequently silver stained. The bands that appeared specifically in the Ctf8-13myc immunoprecipitate were cut out and analyzed by tandem mass spectrometry. Immunoblotting suggests that Ctf8-13myc comigrates with the band indicated with an asterisk, and Ctf8p has run off the gel in the Dcc1-13myc lane. Lane 1: untagged; lane 2: Ctf8-13myc; lane 3: Dcc1-13myc
(B) Immunoprecipitations were performed using equal amounts of protein and either anti-FLAG M2-agarose affinity gel (Sigma) or anti-myc monoclonal antibodies (9E10) and protein A Sepharose CL-4B. Myc-tagged and FLAG-tagged proteins were detected by immunoblotting using anti-myc (9E10) or anti-FLAG (M2; Sigma) monoclonal antibodies. Ctf8p was detected using anti-Ctf8p polyclonal antibodies. CE: cell extract; S: supernatant; P: precipitate
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Probing the Molecular Architecture of the RFC(Ctf18p, Ctf8p, Dcc1p) Complex
Immunoprecipitations were performed from equal amounts of protein using either anti-FLAG M2-agarose affinity gel (Sigma) or anti-myc monoclonal antibodies (9E10) and protein A Sepharose CL-4B. Myc-tagged proteins were detected by immunoblotting using monoclonal anti-myc antibody (9E10). FLAG-tagged proteins were detected using anti-FLAG (M2; Sigma) antibodies, and Ctf8p was detected using anti-Ctf8p polyclonal antibodies. CE: cell extract; S: supernatant; P: precipitate
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 Dcc1p and Ctf18p Are Required for Sister Chromatid Cohesion
(A and B) Wild-type (YPH1466), dcc1Δ (YPH1491), ctf8Δ (YPH1464), ctf18Δ (YPH1492), ctf18Δctf8Δ (YPH1495), ctf18Δdcc1Δ ctf8Δ (YPH1496), and dcc1Δ ctf8Δ (YPH1494) strains were plated on plates containing 0 or 15 μg/ml benomyl. Plates were grown for 3 days at 30°C.
The number of GFP signals was assessed in 100 cells/sample in the following strains:
(C) Wild-type (YPH1477) and two dcc1Δ strains (YPH1487 and YPH1488)
(D) Wild-type (Y819) and two dcc1Δ strains (YPH1499 and YPH1500)
(E) Wild-type (YPH1477) and two ctf18Δ strains (YPH1489 and YPH1490)
(F) Wild-type (YPH1477) and two ctf18Δ ctf8Δdcc1Δ strains (YPH1506 and YPH1507)
The data shown represents the average of two independent experiments
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 Rfc4p Is Required for Sister Chromatid Cohesion
(A) RFC4 (YPH1509) and rfc4-20 (YPH1510) strains were grown at 25°C to early log phase and then shifted to 37°C and allowed to grow for 3 hr. DNA content was measured by FACS.
(B) Sister chromatid cohesion was assessed on chromosome V in RFC4 (YPH1511) and rfc4-20 (YPH1512) strains. The data shown represents the average of two independent experiments. One hundred cells were counted for each sample
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7
The RFC(Ctf18p, Ctf8p, Dcc1p) Complex as a Clamp Loader and Mediator of Polymerase Switching
(A) Alternative RFC complexes as clamp loaders. RFC(Rfc1p) loads PCNA during the process of DNA replication. RFC(Rad24p) is likely to load a clamp-like structure composed of Ddc1p, Rad17p, and Mec3p. RFC(Ctf18p, Ctf8p, Dcc1p) may load PCNA or a currently unidentified clamp.
(B) A model for the role of RFC(Ctf18p, Ctf8p, Dcc1p) in polymerase switching. The replication fork pauses prior to replicating DNA regions that are bound by cohesin. In a competition reaction for binding to RPA, DNA polymerase δ is replaced by RFC(Ctf18p, Ctf8p, Dcc1p), which subsequently loads PCNA or an alternative clamp (data not shown). The RFC(Ctf18p, Ctf8p, Dcc1p) complex is then replaced by DNA polymerase κ, which replicates cohesin-associating regions
Molecular Cell 2001 7, DOI: ( /S (01) )