Structural analysis of the inactive state of the
            <i>Escherichia coli</i>
            DNA polymerase clamp-loader complex

Kazmirski, Steven L.; Podobnik, Marjetka; Weitze, Tanya F.; O'Donnell, Mike; Kuriyan, John

doi:10.1073/pnas.0407904101

Cited by 64 publications

(55 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The C-terminal tetramerization domain is composed of several ␣-helices that pack alongside neighboring subunits to mediate MgsA oligomerization. Overall, the oligomerization domains form a tightly bound collar, and the N-terminal AAA ϩ domains extend out from the collar in a splayed out arrangement, similar to the architecture seen in the inactive state of the E. coli clamp loader (68). The most striking differences between the structure of MgsA and the clamp loader complexes are that MgsA assembles as a tetramer as opposed to the pentameric arrangement observed for the clamp loaders and that the collar domain of MgsA forms a closed oligomer rather than the open arrangement of the clamp loader complex.…”

Section: Resultsmentioning

confidence: 82%

Structure and Biochemical Activities of Escherichia coli MgsA

Page

George

Marceau

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 82%

Structure and Biochemical Activities of Escherichia coli MgsA

Page

George

Marceau

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

“…To define such a small but essential structural change of the clamp loader along with the clamp-loading reactions, further precise structural studies are required. Notably, the binding of two ATP molecules to the E. coli ␥ complex has triggered no significant structural change from the nucleotide-free ''inactive'' complex, suggesting that the binding of the third ATP (the ␥ complex binds up to three ATPs) could cause a structural change of the clamp loader to an active form (27).…”

Section: Resultsmentioning

confidence: 99%

Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis

Miyata

Suzuki

Oyama

et al. 2005

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

111

129

View full text Add to dashboard Cite

Ring-shaped sliding clamps and clamp loader ATPases are essential factors for rapid and accurate DNA replication. The clamp ring is opened and resealed at the primer-template junctions by the ATP-fueled clamp loader function. The processivity of the DNA polymerase is conferred by its attachment to the clamp loaded onto the DNA. In eukarya and archaea, the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) play crucial roles as the clamp loader and the clamp, respectively. Here, we report the electron microscopic structure of an archaeal RFC-PCNA-DNA complex at 12-Å resolution. This complex exhibits excellent fitting of each atomic structure of RFC, PCNA, and the primed DNA. AAA ϩ ATPase ͉ clamp loader ͉ DNA replication ͉ electron microscopy ͉ single-particle analysis I n highly processive genomic DNA duplication, the DNA polymerase is tethered on the DNA strand through a direct interaction with the sliding clamp, which is topologically linked to the DNA by the action of the clamp loader (1). In this reaction, the clamp loader opens and reseals the clamp ring at the primer-template junctions in an ATP-dependent manner. Functional (2-8) and structural (9-12) analyses have indicated that the clamp-loading mechanism is conserved across the domains of life (13-15). All of the sliding clamps from phage to eukarya form similar planer rings, despite their distinct subunit compositions and lower sequence identities. Likewise, the clamp loader complexes from various organisms commonly exist as pentameric complexes with similar subunit configurations. The complexes have a unique oligomeric shape with the open ring in the N-terminal regions of each subunit, which folds into an architecture classified within the AAA ϩ ATPase superfamily (16), while the C-terminal regions form the closed ''collar'' structure. The crystal structure of the yeast clamp loader, replication factor C (RFC), in complex with the sliding clamp, proliferating cell nuclear antigen (PCNA), revealed their detailed contact mode and the elegant match of the spiral configuration of the Nterminal domains of RFC with that of the double-stranded (ds) DNA, and thus allowed the reasonable model building of the RFC-PCNA binary complex docked with a DNA duplex (12).We previously reported the 23-Å resolution EM structure of a clamp-loading RFC-PCNA-DNA ternary complex from Pyrococcus furiosus (Pfu), which was stabilized by introducing a nonhydrolyzable ATP analog, ATP␥S (17). The structure showed the two building blocks, a larger horseshoe and a smaller closed ring. It appeared the best interpretation based on the available data that the horseshoe and the closed ring correspond to RFC and PCNA, respectively. Although the atomic structures of the PCNA trimer (18) and RFC small subunits (RFCSs) (11) were available, along with the information about the 1:4 stoichiometry for RFC large subunit (RFCL) and RFCS in the RFC hetero-pentamer (5), the fitting of the atomic model into the EM map was not completely satisfactory, and some ambiguity remain...

show abstract

“…9), as well as the ␥ subunit adjacent to the ␦ subunit, greatly reduced DNA binding. In a crystal structure of the minimal E. coli clamp loader (␥ 3 ␦␦Ј), the middle ␥ subunit does not contain a bound ATP␥S molecule, whereas the other two ␥ subunits are bound to ATP␥S (37). Biochemical characterization showed that this minimal clamp loader (␥ 3 ␦␦Ј) had greatly reduced DNA binding activity compared with ␥ complex (␥ 3 ␦␦Ј), which is consistent with the idea that ATP binding to the middle ␥ subunit is important for DNA binding (21).…”

Section: Discussionmentioning

confidence: 99%

A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of β Clamp Binding by the Escherichia coli γ Complex Clamp Loader

Thompson

Paschall

O'Donnell

et al. 2009

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

In Escherichia coli, the ␥ complex clamp loader loads the ␤-sliding clamp onto DNA. The ␤ clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, ␥ complex binds ␤ and DNA to form a ternary complex. Binding to primed template DNA triggers ␥ complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of ␥ complex binding ␤ and DNA. A fluorescence intensity-based ␤ binding assay was developed in which the fluorescence of pyrene covalently attached to ␤ increases when bound by ␥ complex. Using this assay, an association rate constant of 2.3 ؋ 10 7 M ؊1 s ؊1 for ␥ complex binding ␤ was determined. The rate of ␤ binding was the same in experiments in which ␥ complex was preincubated with ATP before adding ␤ or added directly to ␤ and ATP. In contrast, when ␥ complex is preincubated with ATP, DNA binding is faster than when ␥ complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote ␤ binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for ␥ complex to bind ␤ before DNA during the clamp loading reaction cycle.Two accessory factors, a sliding clamp and a clamp loader, enhance the efficiency of DNA replication by increasing the processivity of DNA synthesis. The clamp loader assembles clamps onto DNA. The clamp binds the DNA polymerase to increase the processivity of DNA synthesis from tens to thousands of nucleotides in a single binding event. These processivity factors are conserved from bacteria to humans (reviewed in Refs. 1 and 2). The Escherichia coli ␤ clamp is composed of two identical protein monomers assembled into a ring-shaped dimer, which encircles duplex DNA (3, 4). The clamp slides freely along the DNA while tethering DNA polymerase III to the DNA template. The E. coli clamp loader is composed of seven subunits. At the replication fork, a complete clamp loader includes three copies of the dnaX gene product and one copy each of ␦, ␦Ј, , and (5-7). The dnaX gene produces two proteins, a full-length protein () and a truncated protein (␥), which is created by a translational frameshift (8 -10). Clamp loaders containing either three copies of the subunit ( complex, 3 ␦␦Ј) or three copies of the ␥ subunit (␥ complex, ␥ 3 ␦␦Ј) are fully active in clamp loading (6). The subunits have an additional C-terminal extension that coordinates the activities of the replisome (reviewed in Refs. 11 and 12). The clamp loader used in all experiments in this study is the ␥ complex.In the presence of ATP, ␥ complex binds with high affinity to ␤ and DNA to form a ternary complex. Binding to primed template DNA triggers the clamp loader to hydrolyze all three molecules of ATP (...

show abstract

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Cited by 64 publications

References 40 publications

Structure and Biochemical Activities of Escherichia coli MgsA

Structure and Biochemical Activities of Escherichia coli MgsA

Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis

A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of β Clamp Binding by the Escherichia coli γ Complex Clamp Loader

Contact Info

Product

Resources

About