Replication protein A-directed unloading of PCNA by the Ctf18 cohesion establishment complex - PubMed (original) (raw)

Replication protein A-directed unloading of PCNA by the Ctf18 cohesion establishment complex

Göran O Bylund et al. Mol Cell Biol. 2005 Jul.

Abstract

The replication clamp PCNA is loaded around DNA by replication factor C (RFC) and functions in DNA replication and repair. Regulated unloading of PCNA during the progression and termination of DNA replication may require additional factors. Here we show that a Saccharomyces cerevisiae complex required for the establishment of sister chromatid cohesion functions as an efficient unloader of PCNA. Unloading requires ATP hydrolysis. This seven-subunit Ctf18-RFC complex consists of the four small subunits of RFC, together with Ctf18, Dcc1, and Ctf8. Ctf18-RFC was also a weak loader of PCNA onto naked template-primer DNA. However, when the single-stranded DNA template was coated by the yeast single-stranded DNA binding protein replication protein A (RPA) but not by a mutant form of RPA or a heterologous single-stranded DNA binding protein, both binding of Ctf18-RFC to substrate DNA and loading of PCNA were strongly inhibited, and unloading predominated. Neither yeast RFC itself nor two other related clamp loaders, containing either Rad24 or Elg1, catalyzed significant unloading of PCNA. The Dcc1 and Ctf8 subunits of Ctf18-RFC, while required for establishing sister chromatid cohesion in vivo, did not function specifically in PCNA unloading in vitro, thereby separating the functionality of the Ctf18-RFC complex into two distinct paths.

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Figures

FIG. 1.

FIG. 1.

Yeast RFC-like clamp loaders. The indicated complexes (2 μg each) were separated by 10% SDS-PAGE (lanes 1 to 6) or 15% SDS-PAGE (in order to visualize Ctf8;, lane 7), and the gel was stained with colloidal Coomassie. Migration positions of marker proteins are indicated between lanes 6 and 7. RFC-1ΔN (lane 1) lacks a dispensable N-terminal 272-amino-acid domain and was used in most of the studies in this paper (see Materials and Methods).

FIG. 2.

FIG. 2.

The C-terminal tail of Ctf18 is required for binding the Ctf8 and Dcc1 subunits. Lanes 1 to 4 are the input complexes: lane 1, Ctf18-RFC(5); lane 2, Ctf18-ΔC82-RFC(5); lane 3, Rfc2-5; lane 4, His7-Ctf8-Dcc1. Lanes 5 to 8 are the 100 mM imidazole eluates from the Ni2+-agarose column. Lane 5, Ctf18-RFC(5) incubated with His7-Ctf8-Dcc1; lane 6, Ctf18-RFC(5) incubated with His7-Ctf8-Dcc1 and 1 mM ATP; lane 7, Ctf18-ΔC82-RFC(5) incubated with His7-Ctf8-Dcc1 and 1 mM ATP; lane 8, Rfc2-5 incubated with His7-Ctf8-Dcc1 and 1 mM ATP. See Materials and Methods for details.

FIG. 3.

FIG. 3.

RPA inhibits PCNA loading by Ctf18-RFC. (A) Deca-primed ssDNA was coated with RPA where indicated. Loading of 32P-PCNA by RFC (open symbols) or Ctf18-RFC (filled symbols) and ATP where indicated and BioGel-A5m gel filtration were as described in Materials and Methods (see inset for a scheme of the assay). The amount of PCNA (fmol) in each fraction is indicated. The dashed line shows background loading of PCNA by RFC or by Ctf18-RFC without ATP present (results were identical). (B) DNA was incubated with increasing levels of RPA at 30°C for 10 min prior to loading, and gel filtration was done as for (A). The void volume fractions (DNA-PCNA) were acid precipitated and analyzed by SDS-PAGE as described in Materials and Methods. The gel was stained with colloidal Coomassie (upper panel), dried, and subjected to phosphorimager analysis (lower panel). Saturation binding of the ssDNA was achieved with ∼45 pmol of RPA (22). The open arrow to the right indicates a proteolytic fragment of RPA. It migrates close to, but is distinct from, PCNA.

FIG. 4.

FIG. 4.

Unloading of PCNA from DNA by Ctf18-RFC. (A) Scheme of the assay. See Materials and Methods for details. (B) Unloading by Ctf18-RFC and Ctf18-RFC(5). Beads were incubated with Ctf18-RFC without nucleotide, with 1 mM ATP, or with 100 μM ATPγS. Bead-associated radioactivity was determined at the indicated times as described in Materials and Methods. (C) Mutant Ctf18-K189E replaced the wild type in Ctf18-RFC. (D) Successive 10-fold serial dilutions of a ctf18-Δ strain containing either empty vector (−), plasmid pBL441 (CTF18), pBL441-KE (ctf18-K189E), or pBL441-ΔC82 [_ctf18_-Δ(82)] were grown for 3 days at 30°C on yeast-peptone-dextrose plates or yeast-peptone-dextrose plates containing 10 μg/ml of camptothecin. (E) Unloading of PCNA by other clamp loaders as described in Materials and Methods. The assays were carried out in duplicate in two or three independent assays, and the average is given. Standard errors ranged from 5 to 10% for all assays.

FIG. 5.

FIG. 5.

Rates of PCNA unloading by Ctf18-RFC. (A) Scheme of the assay. Multiple 32P-PCNA rings were loaded onto SKII+ DNA containing a 90-nt ssDNA gap. (B) 32P-PCNA was loaded onto gapped DNA by RFC and isolated by BioGel-A5m filtration as described in Materials and Methods. Isolated complex containing 120 fmol 32P-PCNA was incubated for 5 min at 20°C with 250 fmol of Ctf18-RFC where indicated and either without nucleotide or with 1 mM ATP or 100 μM ATPγS. After glutaraldehyde cross-linking and gel electrophoresis, the gel was dried and subjected to phosphorimager analysis. Migration positions of glutaraldehyde-cross-linked PCNA and Ctf18-RFC-PCNA were determined independently. (C) Unloading reactions were identical to those analyzed in (B), except that increasing amounts of Ctf18-RFC were used, and aliquots were removed at the indicated times and cross-linked prior to electrophoresis. The DNA-PCNA band shown in (B) was quantitated.

FIG. 6.

FIG. 6.

Unloading of PCNA by RFC and Ctf18-RFC. (A) 32P-PCNA was loaded onto nicked DNA by RFC and isolated by BioGel-A5m filtration as described for Fig. 5A and in Materials and Methods. Isolated complex containing 3 nM 32P-PCNA was incubated at 20°C with 1 mM ATP and either no complex, 10 nM of Ctf18-RFC, or 10 nM of wild-type RFC. Aliquots were taken after 15 s, 60 s, 3 min, and 10 min, cross-linked with glutaraldehyde, and further processed for analysis. (B) unloading of PCNA from nicked DNA. Assays were similar to those in (A), and the same time points were used. Experiment 1, 0.3 nM DNA-PCNA and 0.5 nM Ctf18-RFC; experiment 2, 0.3 nM DNA-PCNA and 0.5 nM wild-type RFC; experiment 3, 3 nM DNA-PCNA and 10 nM RFC-1ΔN; experiment 4, 3 nM DNA-PCNA and 10 nM Ctf18-RFC previously incubated with a twofold molar excess of PCNA for 30 min at 0°C. (C) Unloading of PCNA from RPA-coated deca-primed SS DNA (see Fig. 3A). Assays were carried out as in (B): experiment 5, 3 nM DNA-PCNA and 10 nM Ctf18-RFC; experiment 6, 3 nM DNA-PCNA and 10 nM Ctf18-RFC previously incubated with a twofold molar excess of PCNA for 30 min at 0°C.

FIG. 7.

FIG. 7.

Possible roles for PCNA loading and unloading in the establishment of sister chromatid cohesion. See the text for details.

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