Mechanism of ATP-Driven PCNA Clamp Loading by S. cerevisiae RFC

Chen, Siying; Levin, Mikhail K.; Sakato, Miho; Zhou, Yayan; Hingorani, Manju

doi:10.1016/j.jmb.2009.03.014

Cited by 51 publications

(110 citation statements)

References 34 publications

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“…This enzyme superfamily has functionally diverse roles that include not only RNA and DNA helicases, proteases, chaperones, and metal chelatases but many additional DNA replication factors such as processivity clamp loaders (e.g., replication factor C [38] and the ␥ complex [116]), initiator proteins (e.g., DnaA and the origin recognition complex [72]), and helicase loaders (e.g., DnaC [57] and Cdc6 [152]). The Mcm proteins, in common with many members of this group, oligomerize into ring-shaped (i.e., toroidal) complexes (23,193) and use ATP binding and hydrolysis to manipulate a substrate (e.g., DNA) within their central channels.…”

Section: The Eukaryotic MCM Genes Define Six Families Of Aaa ؉ Motor mentioning

confidence: 99%

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

Bochman

Schwacha

2009

Microbiol Mol Biol Rev

293

278

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SUMMARY The Mcm2-7 complex serves as the eukaryotic replicative helicase, the molecular motor that both unwinds duplex DNA and powers fork progression during DNA replication. Consistent with its central role in this process, much prior work has illustrated that Mcm2-7 loading and activation are landmark events in the regulation of DNA replication. Unlike any other hexameric helicase, Mcm2-7 is composed of six unique and essential subunits. Although the unusual oligomeric nature of this complex has long hampered biochemical investigations, recent advances with both the eukaryotic as well as the simpler archaeal Mcm complexes provide mechanistic insight into their function. In contrast to better-studied homohexameric helicases, evidence suggests that the six Mcm2-7 complex ATPase active sites are functionally distinct and are likely specialized to accommodate the regulatory constraints of the eukaryotic process.

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Section: The Eukaryotic MCM Genes Define Six Families Of Aaa ؉ Motor mentioning

confidence: 99%

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

Bochman

Schwacha

2009

Microbiol Mol Biol Rev

293

278

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“…The clamp is loaded onto DNA by the clamp loader, a five-subunit complex of AAAϩ family proteins that couple ATP binding and hydrolysis to mechanical work (8 -13). Structural and mechanistic analyses of clamp loaders such as E. coli ␥ complex (11,14), bacteriophage T4 gp44/62 (8,15,16), and Saccharomyces cerevisiae RFC (10,(17)(18)(19) among others, have shown that ATP binding enables the clamp loader to bind and open the clamp and bind ptDNA, and ATP hydrolysis leads to the release of the clamp-ptDNA product (see Fig. 1A), which can then be used by DNA polymerase and other proteins.…”

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“…1A), which can then be used by DNA polymerase and other proteins. In recent pre-steady state kinetic studies of S. cerevisiae RFC and PCNA, we explicitly measured the rates of individual steps in the reaction, including PCNA opening/closing, ptDNA binding/release, ATP hydrolysis, and phosphate (P i ) release (17,18). The data revealed a pair of slow events that bookend the loading reaction: the first occurs after ATP binding to RFC (RFC acti-vation) and involves conformational changes that enable PCNA opening and ptDNA binding to form an RFC-ATP-PCNA openptDNA intermediate primed for ATP hydrolysis; the second occurs after ATP hydrolysis (RFC deactivation) and involves conformational changes that enable PCNA closure and release of the PCNA-ptDNA product.…”

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confidence: 99%

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Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

Zhou

Hingorani

2012

Journal of Biological Chemistry

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Background: Crystal structures reveal direct clamp-DNA contacts whose functions are unclear. Results: Mutations of single N-terminal arginines/lysines in the inner PCNA ring affect DNA binding-linked steps in the clamp loading mechanism. Conclusion: Loss of individual PCNA-DNA contacts disrupts clamp loading and its interaction with DNA. Significance: DNA binding to specific locations within circular clamps influences other proteins that work with clamps on DNA.

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“…In the presence of ATPγS the RFC · PCNA complex opened the clamp and bound to DNA but ring closure was not completed (19,20). Pre-steady-state global analysis of the accompanying ATP hydrolysis showed that the ATPase reaction proceeded through multiple steps within two distinct phases; first, assembly of the RFC · PCNA · DNA complex was coupled to ATP binding and second disassembly was coupled to ATP hydrolysis (21). Completion of a functioning holoenzyme requires addition of polδ to the RFC · PCNA · DNA complex.…”

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confidence: 99%

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

Kumar

Nashine

Mishra

et al. 2010

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

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In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC · PCNA · DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC · PCNA · DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC · PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polδ only one species converts to the RFC · PCNA · DNA · polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.DNA replication | replication factor C | single-molecule FRET D NA replication is carried out by effective coordination of the activities of many proteins at the replication fork. Given the complex nature of DNA synthesis, a structural and functional conservation of the accessory proteins apparently has evolved across diverse organisms (1-3). However, eukaryotic DNA replication has diverged and become more complicated than its prokaryotic counterpart due in part to the multisubunit composition of several proteins. In yeast, lagging strand DNA synthesis requires the coordinated actions of polymerase δ (polδ), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) (4). Eukaryotic PCNA, a ring-shaped trimer of identical subunits, plays a pivotal role in both DNA replication and repair by tethering the DNA polymerase to significantly enhance the processivity of DNA synthesis (5-10). The productive assembly of a polymerase-PCNA complex bound to DNA requires loading of the PCNA clamp by RFC (11). RFC, an ATP-driven motor, is composed of a large subunit (RFC-A, 95 kDa) and four small subunits (RFC-B-RFC-E, 36-40 kDa) and forms a RFC · PCNA · ATP complex that binds specifically to primed DNA (12).The clamp-loading process occurs via multiple steps (13-16). X-ray crystallographic and electron microscopic studies of the clamp loader complexed with the clamp revealed that PCNA was either in a closed or slightly open conformation that perhaps represents the structure of an intermediate step in PCNA loading (13,17). PCNA, in solution, has a closed conformation and must be opened for loading on DNA and further reclosed to be retained on the DNA possibly through a specific single interface (18).The structure of the yeast RFC · PCNA complex (13) provided the coordinates for introducing a fluorescent donor-acceptor pair within the PCNA to probe the FRET efficiencies distance information on the conformation of the PCNA subunit interface (19). In the presence of ATP, the PCNA...

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Mechanism of ATP-Driven PCNA Clamp Loading by S. cerevisiae RFC

Cited by 51 publications

References 34 publications

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

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