The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA

Abstract

The linkage of sister chromatids after DNA replication ensures the faithful inheritance of chromosomes by daughter cells. In budding yeast, the establishment of sister chromatid cohesion requires Ctf8, Dcc1, and Ctf18, a homologue of the p140 subunit of the replication factor C (RFC). In this report we demonstrate that in 293T cells, Flag-tagged Ctf18 forms a seven-subunit cohesion-RFC complex comprised of Ctf18, Dcc1, Ctf8, RFCp40, RFCp38, RFCp37, and RFCp36 (Ctf18-RFC). We demonstrate that a stoichiometric heteroheptameric Ctf18-RFC complex can be assembled by coexpressing the seven proteins in baculovirus-infected insect cells. In addition, the two other stable subcomplexes were formed, which include a pentameric complex comprised of Ctf18, RFCp40, RFCp38, RFCp37, and RFCp36 and a dimeric Dcc1-Ctf8. Both the five- and seven-subunit Ctf18-RFC complexes bind to single-stranded and primed DNAs and possess weak ATPase activity that is stimulated by the addition of primed DNA and proliferating cell nuclear antigen (PCNA). These complexes catalyzed the ATP-dependent loading of PCNA onto primed and gapped DNA but not onto double-stranded nicked or single-stranded circular DNAs. Consistent with these observations, both Ctf18-RFC complexes substituted for the replicative RFC in the PCNA-dependent DNA polymerase delta-catalyzed DNA replication reaction. These results support a model in which sister chromatid cohesion is linked to DNA replication.

Fig. 1.

Isolation of Ctf18-RFC complexes. (A) Isolation of Ctf18 complexes from 293T cells. A silver-stained SDS/12% polyacrylamide gel of the Flag peptide eluted Ctf18 complex (0.46 μg of protein) isolated from stably transfected 293T cells as described in Materials and Methods is shown. All proteins indicated were identified by mass spectrometry with the exception of Ctf8, which was detected by Western blot analysis. (B) Characterization of Ctf complexes isolated from baculovirus-infected Sf9 cells. Shown is a Coomassie-stained SDS/12% polyacrylamide gel of purified Ctf complexes isolated by glycerol gradient centrifugation as described in Materials and Methods.(Left) Containing 2 μg of purified Ctf18-RFC(7S). (Center) Containing 1.2 μg of Ctf18-RFC(5S). (Right) Containing 6 μg of Dcc1-Ctf8 heterodimer.

Fig. 2.

Order of assembly of the Ctf18-Dcc1-Ctf8-RFC2-5 complex. An IVT system containing the indicated expression vectors was used to synthesize labeled proteins. Products were immunoprecipitated with the antibody indicated above the lanes, and the precipitated material then was separated by SDS/PAGE and analyzed by autoradiography. (A) Interaction of Ctf18, Dcc1, or Ctf8 with RFC2-5. (B) Critical role of the RFC5(p38) subunit in the formation of the Ctf18-RFC2-5 complex. Flag-tagged Ctf18 complexes were adsorbed to Flag-M2 antibody beads. + and -, presence or absence, respectively, of the competing Flag peptide (1 mg) added in the immunoprecipitation step. (C) Formation of six- and seven-subunit Ctf18-RFC complexes. (D) Interactions among the Ctf18, Dcc1, and Ctf8 subunits. LO, load-on; PR, preimmune serum; D1, Dcc1-specific antibodies; C8, Ctf8-specific antibodies.

Fig. 3.

Competition of the binding of the Ctf18-RFC(7S) to labeled primed DNA by unlabeled DNAs. Reaction mixtures (15 μl) containing 0.5 pmol of clamp loader, 100 fmol of ³²P-labeled primed 50-bp dsDNA with a 50-nt ssDNA (5′ overhang), unlabeled DNAs as indicated, 25 mM Hepes (pH 7.5), 3 mM MgCl2, 1 mM DTT, and 0.1 mg/ml BSA were incubated for 30 min on ice. The DNA bound to the protein complex was quantified by a nitrocellulose filter-binding assay. The DNA substrate used has been described (40).

Fig. 4.

Loading of ³²P-labeled PCNA onto indicated DNA templates by the five- and seven-subunit Ctf18 complexes. Reactions (50 μl) contained 150 fmol of indicated DNA substrate, 0.6 pmol of ³²P-labeled PCNA, 0.2 pmol of PCNA clamp loader, 5.71 μg of hRPA, 40 mM Tris·HCl (pH 7.8), 10 mM magnesium acetate, 200 μg/ml BSA, and 50 mM NaCl. After 10 min at 37°C, the mixtures were loaded immediately onto a 5-ml Bio-Rad A-15 column equilibrated with the reaction buffer containing 150 mM NaCl. The distribution of the [³²P]PCNA in eluted fractions was monitored by Cerenkov counting. (A) Loading of PCNA onto hexaprimed M13 ssDNA by the five- and seven-subunit Ctf18-RFC complex in the presence and absence of ATP. (B)Influence of NTPs on the loading of PCNA onto hexaprimed M13 DNA by Ctf18-RFC(7S). (C) Loading of PCNA by Ctf18-RFC(7S) on RPA-coated hexaprimed M13 DNA (M13-RPA), E. coli SSB-coated hexaprimed M13 DNA (M13-SSB), RPA-coated M13 DNA (ssM13-RPA), and nicked pBluescript (nick-ATP). (D) Loading of PCNA by RFC on RPA-coated hexaprimed M13 DNA (M13) or nicked pBluescript (nick) in the presence and absence of ATP.

Fig. 5.

Cohesion RFC supports the Pol δ-catalyze elongation of primed DNA. Reaction mixtures were as described (41) and contained 10 fmol of singly primed M13 ssDNA coated with 2.5 pmol of hRPA or E. coli SSB, 0.2 pmol of hPCNA, 100 fmol of hPol δ, and indicated levels of RFC or Ctf18 RFC. After 30 min at 37°C, aliquots of reaction mixtures were subjected to alkaline agarose electrophoresis and used to measure DNA synthesis. The nucleotide incorporation in each reaction is indicated below each lane. The position of fully extended DNA chains is indicated by the 7-kb marker.

Fig. 6.

Coimmunoprecipitation of Scc1 and SMC1 with Ctf18 from stably transfected 293T cells. The cell pellet (8 ml) was lysed in 25 ml of lysis buffer as described in Materials and Methods. The mixture was sonicated, digested with 250 μg of pancreatic DNase I overnight on ice, and then centrifuged at 34,500 × g for 30 min at 4°C. An aliquot of the supernatant (12.5 ml, 125 mg of protein) was incubated with 200 μl of preequilibrated Flag beads or Myc beads (negative control) at 4°C overnight. Beads then were washed six times with 1 ml of PBS containing 0.05% Nonidet P-40, 1 mM DTT, and proteinase inhibitors, and the bound Flag-tagged Ctf18 complex was eluted with 1 mg/ml Flag₃ peptide (Flag El) or with 1 mg/ml Myc peptide as indicated. The headings Ttl, Sol, and FT refer to total lysate, load-on (solubilized material), and material that did not bind (flow-through), respectively. Approximately 30 μg of protein of the Ttl, Sol, and FT fractions and 0.5 μg of the material eluted with the Flag peptide from Flag beads (Flag-El) and Myc beads (Myc-El) were loaded onto a SDS/12% PAGE followed by Western blotting with the antibodies specific for Ctf18, SMC1, and Scc1 (45).