Overproduction and Affinity Purification of Saccharomyces cerevisiae Replication Factor C

Gerik, Kimberly J.; Gary, Sonja L.; Burgers, Peter M.

doi:10.1074/jbc.272.2.1256

Cited by 73 publications

(79 citation statements)

References 25 publications

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“…Rad17/3/1 and RFCRad24 interact in the absence of DNA (6,9,10). As previously observed with PCNA and RFC, the formation of a stable Rad17/ 3/1⅐RFC-Rad24 complex was strongly enhanced by ATP binding but did not require its hydrolysis (12). Loading of the Rad17/3/1 clamp around partial duplex DNA can be mediated by either ATP or ATP␥S; however, ATP hydrolysis was required to release the clamp from the clamp loader and permit sliding of Rad17/3/1 across double-stranded DNA (8,10).…”

supporting

confidence: 58%

Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

Majka

Chung

Burgers

2004

Journal of Biological Chemistry

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The DNA damage clamp loader replication factor C (RFC-Rad24) consists of the Rad24 protein and the four small Rfc2-5 subunits of RFC. This complex loads the heterotrimeric DNA damage clamp consisting of Rad17, Mec3, and Ddc1 (Rad17/3/1) onto partial duplex DNA in an ATP-dependent manner. Interactions between the clamp loader and the clamp have been proposed to mirror those of the replication clamp loader RFC and the sliding clamp proliferating cell nuclear antigen (PCNA). In that system, three ATP molecules bound to the Rfc2, Rfc3, and Rfc4 subunits are necessary and sufficient for efficient loading of PCNA, whereas ATP binding to Rfc1 is not required. In contrast, in this study, we show that mutant RFC-Rad24 with a rad24-K115E mutation in the ATP-binding domain of Rad24 shows defects in the ATPase of the complex and is defective for interaction with Rad17/3/1 and for loading of the checkpoint clamp. A similar defect was measured with a mutant RFCRad24 clamp loader carrying a rfc4K55R ATP-binding mutation, whereas the rfc4K55E clamp loader showed partial loading activity, in agreement with genetic studies of these mutants. These studies show that ATP utilization by the checkpoint clamp/clamp loader system is effectively different from that by the structurally analogous replication system.

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confidence: 58%

Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

Majka

Chung

Burgers

2004

Journal of Biological Chemistry

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“…In this case, the RFC(3/4 SAC ) mutant should still be stimulated by RFC2 to the same extent as wild-type RFC complex. If RFC2 were to bind RFC4, instead of RFC3, addition of RFC2 would not stimulate the activity of the resulting RFC(2/3/4 SAC ) com- RFC (3/4) and RFC(2/3) Bind PCNA-RFC, like prokaryotic ␥ complex, binds the clamp in an ATP-dependent fashion (30). ATP binding is sufficient to promote the RFC⅐PCNA complex, hydrolysis is not required.…”

Section: Resultsmentioning

confidence: 99%

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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“…6B, lanes 2,6). To further characterize the interaction, the stable Rfc2-5 subcomplex (Gerik et al 1997) was purified and tested for association with Asf1 to determine whether the large subunit of RFC (Rfc1) was necessary for the interaction. Like RFC, Rfc2-5 coprecipitated in an Asf1-dependent manner (Fig.…”

Section: Asf1 Interacts With Rfcmentioning

confidence: 99%

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

Franco¹,

Lam²,

Burgers³

et al. 2005

Genes Dev.

128

125

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Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase (Pol ), are reduced at stalled replication forks. In contrast, DNA polymerase ␣ (Pol ␣) accumulates to higher than normal levels at stalled forks in asf1⌬ cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.[Keywords: Chromatin assembly; histone deposition; DNA replication; genome stability] Supplemental material is available at http://www.genesdev.org. In eukaryotes, DNA is assembled into a nucleoprotein complex called chromatin. The fundamental repeating unit of chromatin is the nucleosome, which consists of 146 bp of DNA wrapped around an octamer of core histone proteins, comprised of two H2A/H2B dimers flanking an inner (H3/H4) 2 tetramer. In vivo, histone deposition can occur by DNA replication-coupled or replication-independent mechanisms (for review, see Franco and Kaufman 2004). In either case, histone deposition is mediated by specialized assembly proteins. The best characterized replication-coupled chromatin assembly factor is chromatin assembly factor-1 (CAF-1), an evolutionarily conserved protein complex that binds histone H3 and H4 and delivers them to replicating DNA through an interaction with proliferating cell nuclear antigen (PCNA), the DNA polymerase processivity protein (for review, see Asf1, another evolutionarily conserved histone chaperone, functions during both replication-coupled and replication-independent chromatin assembly. Asf1 bound to histones H3/H4 stimulates histone deposition by CAF-1 in vitro (Tyler et al. 1999;Sharp et al. 2001), and Asf1 physically interacts with the p60/Cac2 subunit of CAF-1 (Tyler et al. 2001;Krawitz et al. 2002;Mello et al. 2002). In yeast, Asf1 and the Hir proteins directly interact and function together to promote heterochromatic gene silencing (Kaufman et al. 1998;Sharp et al. 2001;Sutton et al. 2001;Krawitz et al. 2002). Consistent with a role in multiple deposition pathways, Asf1 copurifies with both CAF-1 and Hir protein complexes in human cell extracts (Tagami et al. 2004).Asf1 also contributes to genome stability during S phase in a manner distinct from both CAF-1 and the Hir proteins. Unlike yeast cells lacking CAF-1 or Hir proteins, cells lacking Asf1 are sensitive to the DNA synthesis inhibitor hydroxyurea (HU) and the DNA topoisomerase I inhibitor camptothecin (CPT), and display synthetic genetic interactions with DNA synthesis genes including the initiation/elongation factor C...

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Overproduction and Affinity Purification of Saccharomyces cerevisiae Replication Factor C

Cited by 73 publications

References 25 publications

Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

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

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

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