Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells

Cai, Jinsong; Uhlmann, Frank; Gibbs, E. Michael; Flores‐Rozas, Hernan; Lee, Chee-Gun; Phillips, Barbara B.; Finkelstein, Jeff; Yao, Nina Y.; O'Donnell, Mike; Hurwitz, Jerard

doi:10.1073/pnas.93.23.12896

Cited by 82 publications

(105 citation statements)

References 47 publications

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“…Both PCNA and 9-1-1 clamps stimulate the DNA-dependent ATPase activity of their clamp-loading complexes (36,42,44). Similar experiments with Ctf18-RFC(7S) demonstrated that PCNA stimulated this activity only Ϸ1.5-fold, whereas 9-1-1 had no effect.…”

Section: Dna-binding Properties Of the Seven-and Five-subunit Ctf18 -Rfcsupporting

confidence: 51%

“…The DNA-binding properties of replicative RFC and checkpoint Rad17-RFC are similar (42,43). In the presence of 175 mM NaCl, both complexes bind specifically to the 3Ј end of a primed DNA, whereas at 50 mM NaCl they bind to singlestranded (ss) and double-stranded (ds) DNA as well.…”

Section: Dna-binding Properties Of the Seven-and Five-subunit Ctf18 -Rfcmentioning

confidence: 86%

“…These assays were carried out as described (40)(41)(42). Specific additions are indicated in the figure legends.…”

Section: Methodsmentioning

confidence: 99%

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

Bermudez

Maniwa

Tappin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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120

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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 sevensubunit 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 ␦-catalyzed DNA replication reaction. These results support a model in which sister chromatid cohesion is linked to DNA replication.F aithful inheritance of a complete set of the chromosome complement by daughter cells is essential for cell survival (1-4). In eukaryotes, newly synthesized sister chromatid DNAs are linked together physically by the cohesin complex (called cohesion) from the time they are replicated until their distribution between daughter cells in anaphase (1, 5-7). In budding yeast, the cohesin complex, composed of the four proteins Scc1͞Rad21, Scc3, SMC1, and SMC3, is loaded onto chromatin during the G 1 ͞S transition and leads to the association of sister chromatids after DNA replication. Mutations in any one of these subunits result in the precocious separation of sister chromatids before cohesion is severed in anaphase and ultimately leads to cell death. Recent studies have revealed important insights into the cohesin structure and have shown that the severance of cohesion is mediated by separase, a protease that cleaves Scc1 (1,5,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The steps leading to cohesion between sister chromatids, however, remain unknown. Chromosome-loss assays have identified a number of genes required for cohesion of sister chromatids. Based on their putative roles, cohesion genes have been characterized as either deposition or establishment factors (18). The deposition factors such as Scc2 and Scc4, which interact and must function during S phase, are required for the loading of cohesin onto DNA but are not part of the cohesin complex (19). Establishment factors, which include Ctf7͞Eco1͞Eso1, Ctf4͞Pob1, Ctf18͞Chl12, Dcc1, and Ctf8, are essential for cohesion and have some ...

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Section: Dna-binding Properties Of the Seven-and Five-subunit Ctf18 -Rfcsupporting

confidence: 51%

Section: Dna-binding Properties Of the Seven-and Five-subunit Ctf18 -Rfcmentioning

confidence: 86%

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

Bermudez

Maniwa

Tappin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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120

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“…S3), where DL, DL2-L, and DL2-R gave comparable amounts of full-length products, indicating that PCNA was loaded onto these substrates with similar efficiency. All these data indicate that PCNA loading on the DL was not dependent on a 3′ junction, in contrast to the previous studies using primed ssDNA substrates (30)(31)(32)(33). Therefore, RPA most likely binds to the displaced ssDNA loop to stimulate the PCNA loading reaction.…”

contrasting

confidence: 53%

“…PCNA is loaded onto DNA by the "clamp-loader" Replication Factor C (RFC; complex of Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5), which opens and reseals the PCNA on dsDNA in an ATPdependent manner (30)(31)(32)(33). Loaded PCNA encircles and slides freely along the DNA, anchoring a DNA polymerase to the DNA for processive DNA synthesis (34)(35)(36).…”

mentioning

confidence: 99%

PCNA is efficiently loaded on the DNA recombination intermediate to modulate polymerase δ, η, and ζ activities

Holzschu

Sugiyama

2013

Proc. Natl. Acad. Sci. U.S.A.

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Proliferating cell nuclear antigen (PCNA) is required for DNA homologous recombination (HR), but its exact role is unclear. Here, we investigated the loading of PCNA onto a synthetic D-loop (DL) intermediate of HR and the functional interactions of PCNA with Rad51 recombinase and DNA polymerase (Pol) δ, Pol η, and Pol ζ. PCNA was loaded onto the synthetic DL as efficiently as it was loaded onto a primed DNA substrate. Efficient PCNA loading requires Replication Protein A, which is associated with the displaced ssDNA loop and provides a binding site for the clamp-loader Replication Factor C. Loaded PCNA greatly stimulates DNA synthesis by Pol δ within the DL but does not affect primer recognition by Pol δ. This suggests that the essential role of PCNA in HR is not recruitment of Pol δ to the DL but stimulation of Pol δ to displace a DNA strand during DL extension. Both Pol η and Pol ζ extended the DL more efficiently than Pol δ in the absence of PCNA, but little or no stimulation was observed in the presence of PCNA. Finally, Rad51 inhibited both the loading of PCNA onto the DL and the extension of the DL by Pol δ and Pol η. However, preloaded PCNA on the DL counteracts the Rad51-mediated inhibition of the DL extension. This suggests that the inhibition of postinvasion DNA synthesis by Rad51 occurs mostly at the step of PCNA loading.DNA repair | translesion polymerase | sliding clamp D NA double-strand breaks (DSBs) are introduced into the genome by several factors, including ionizing radiation, mutagenic chemicals, reactive oxygen species, and stalled DNA replication (1). Without appropriate repair, DSBs may lead to cell lethality or cancer (2-4). Homologous recombination (HR) is a widely conserved essential mechanism for high-fidelity repair of DSBs (5-8). HR is a highly coordinated multistep biochemical process, which is most elegantly demonstrated in the yeast Saccharomyces cerevisiae. Briefly, DSB ends are first processed by specialized exonucleases to generate 3′ overhangs (9-11), which are then coated with the ssDNA binding protein Replication Protein A (RPA). The Rad52 recombination mediator interacts with RPA and recruits Rad51 recombinase onto the ssDNA to form a helical nucleoprotein filament (12-15). This Rad51-ssDNA filament mediates strand invasion into a homologous dsDNA (16) to produce a DNA structure that is referred to as the D-loop (DL; see Fig. 6B). The 3′ end of the invading strand is used as the primer by DNA polymerases for DNA synthesis (postinvasion DNA synthesis), extending the DL (see Fig. 6F). After postinvasion DNA synthesis, repair may be completed by a break-induced replication, DSB repair, or synthesis-dependent strand-annealing pathway (5).Postinvasion DNA synthesis is crucial to high-fidelity repair of DSBs because it recovers the genetic information that might be lost during breakage events. Several DNA polymerases, including polymerase (Pol) δ, Pol η, and Pol ζ, are possibly involved in this process. Pol δ is known as a replicative polymerase that constitutes the replisome (17,...

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Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus*

et al. 2001

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We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase delta, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.

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Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells

Cited by 82 publications

References 47 publications

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

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

PCNA is efficiently loaded on the DNA recombination intermediate to modulate polymerase δ, η, and ζ activities

Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus*

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