In vitro reconstitution of human replication factor C from its five subunits.

Uhlmann, Frank; Cai, Jinsong; Flores‐Rozas, Hernan; Dean, Frank B.; Finkelstein, Jeff; O’Donnell, Michael; Hurwitz, Jerard

doi:10.1073/pnas.93.13.6521

Cited by 55 publications

(87 citation statements)

References 32 publications

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“…The final arrangement of RFC, illustrated in Fig. 9, confirms earlier proposals based on homologies between RFC and ␥ complex (9, 33) and on previous protein-protein interaction studies of human RFC (28,29).…”

Section: Resultssupporting

confidence: 88%

“…Results of this study are consistent with this assignment as they demonstrate that RFC2, 3, and 4 bind one another and contain an inherent DNA-stimulated ATPase activity. Likewise, the analogous human RFC p40/p37/p36 complex contains DNA-dependent ATPase activity (28,29). Moreover, the current study also confirms the hypothesis that these RFC subunits require the SRC arginine finger for ATP hydro- …”

Section: Resultssupporting

confidence: 85%

“…Hence, we were unable to demonstrate directly that RFC1 is adjacent to RFC5 and RFC4. However, protein-protein interaction studies of human RFC have demonstrated that RFC1 (p140) binds to both RFC4 (p40) and RFC5 (p38) subunits (28,29) thus supporting the above position assigned to RFC1.…”

Section: Resultsmentioning

confidence: 85%

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Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

Yao

Coryell

Zhang

et al. 2003

Journal of Biological Chemistry

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Replication factor C (RFC) is a heteropentameric AAA؉ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, ␥ complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA؉ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli ␥ complex in which only one subunit makes strong contact with the ␤ clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric ␤ ring.Replicases of cellular chromosomes utilize a circular sliding clamp protein that encircles DNA and tethers the polymerase to the template for high processivity in DNA synthesis (1). An example of this protein class is the Escherichia coli ␤ subunit, which confers high processivity onto the chromosomal replicase, DNA polymerase III holoenzyme (2, 3). The eukaryotic equivalent is the PCNA 1 ring, which has essentially the same shape and chain fold as ␤ despite lack of sequence similarity between the two (4, 5). These ring-shaped proteins require an ATP-fueled multiprotein clamp loader for assembly onto primed DNA.The eukaryotic clamp loader is the heteropentameric replication factor C (RFC). The five subunits of RFC are each different proteins, but they are homologous to one another (6, 7) and are members of the AAAϩ family of ATPases (8). The recent crystal structure of E. coli ␥ complex, the prokaryotic counterpart of RFC, has facilitated detailed hypothesis regarding RFC structure and mechanism (9, 10). The ␥ complex (␥ 3 ␦␦Ј ) consists of a minimal core of five proteins (␥ 3 ␦␦Ј), which contain the clamp loading activity (11). The remaining two subunits, and , are involved in recruiting an RNA primed DNA site from the primase, and they bind singlestranded DNA-binding protein (SSB) to assist polymerase elongation but are not essential to the clamp loading activity of ␥ complex (12)(13)(14). Biochemical studies of ␥ complex (15-17), combined with crystal structures of ␥ 3 ␦␦Ј (10) and ␦-␤ 1 complex ...

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Section: Resultssupporting

confidence: 88%

Section: Resultssupporting

confidence: 85%

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Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

Yao

Coryell

Zhang

et al. 2003

Journal of Biological Chemistry

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“…Coupled IVT reactions and immunoprecipitation were performed as described (39). The rabbit antisera used for immunoprecipitations were raised against bacterially expressed hCtf18 (amino acids 1-243), hDcc1, and hCtf8 purified by nickel-chelate chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…The rabbit antisera used for immunoprecipitations were raised against bacterially expressed hCtf18 (amino acids 1-243), hDcc1, and hCtf8 purified by nickel-chelate chromatography. The antiserum against the 37 RFC subunit has been described (39).…”

Section: Methodsmentioning

confidence: 99%

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.

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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Clamp Loaders, Processivity Complex

Kelman¹

2002

Encyclopedia of Molecular Biology

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In vitro reconstitution of human replication factor C from its five subunits.

Cited by 55 publications

References 32 publications

Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

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

Clamp Loaders, Processivity Complex

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