Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

Trakselis, Michael A.; Alley, Stephen C.; Abel-Santos, Ernesto; Benkovic, Stephen J.

doi:10.1073/pnas.111006698

Cited by 104 publications

(103 citation statements)

References 62 publications

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“…Fluorescent energy transfer studies in the T4 phage replication system indicate that the gp45 clamp protomers open out-of-plane with a left-handed helical pitch (20). In the E. coli and S. cerevisiae systems, the right-handed helical arrangement of ␥ 3 ␦␦Ј and RFC subunits is compatible with a righthanded opening of the clamp, thereby allowing it to dock onto the other clamp loading subunits.…”

Section: The Open Clamp Is a Spiral Lockwashermentioning

confidence: 84%

Replisome Architecture and Dynamics in Escherichia coli

O'Donnell

2006

Journal of Biological Chemistry

126

114

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The field of DNA replication was launched upon discovery of the first DNA polymerase by Kornberg and Lehman (1,2). DNA polymerase I displayed the novel and highly exciting property of template-directed enzymatic action, and like a split personality, DNA polymerase I could also degrade DNA, from either direction. Escherichia coli is now known to contain five different DNA polymerases. The chromosomal replicase is DNA polymerase III, while the damage-inducible DNA polymerases II, IV, and V play roles in DNA repair. DNA polymerase I is involved in both replication and repair and remains the most advanced model for the structure and function of DNA polymerase action.DNA polymerase III (pol III) 2 functions in the context of a multiprotein apparatus called DNA pol III holoenzyme (reviewed in (3-5)). pol III holoenzyme contains 10 different proteins, which assort into three major functional units: 1) pol III core, 2) the ␤ sliding clamp, and 3) the ␥/ complex clamp loader. The holoenzyme contains two copies of the pol III core, which are connected by the attachment to one clamp loader. The holoenzyme functions within the context of a dynamic replisome containing pol III holoenzyme, a hexameric DnaB helicase, DnaG primase, and SSB. During replisome function, contacts between these proteins are in a constant state of change. This review briefly summarizes the architecture and dynamic behavior of the E. coli replisome. The Clamp and Clamp LoaderThe ␤ sliding clamp is a homodimer in the shape of a ring, which encircles DNA (Fig. 1A). The ␤ clamp slides on DNA and binds the pol III core, thereby acting as a mobile tether and converting the normally distributive pol III core into a highly processive and rapid polymerase capable of incorporating 500 -1,000 nucleotides per second (Fig. 1C). The crystal structure of the ␤ dimer reveals a 6-fold pseudo symmetry that arises from a domain that is repeated three times in the monomer giving the dimer a 6-fold appearance (6). The eukaryotic PCNA clamp and phage T4 gp45 clamp are also six-domain rings, but the monomeric unit contains only two domains and trimerizes to form a six-domain ring (7).Clamps do not self-assemble onto DNA but require a multiprotein clamp loader, which harnesses the energy of ATP hydrolysis to open and close the clamp around DNA (Fig. 1B). The E. coli ␥ complex clamp loader contains five subunits that are essential for clamp loading activity, three ␥ protomers and one copy each of ␦ and ␦Ј. The small and subunits are not required for clamp loading, but they stabilize the clamp loader and stimulate its activity at elevated ionic strength.The ␥, ␦, and ␦Ј clamp loading subunits are members of the AAAϩ family (ATPases associated with a variety of cellular functions) (8). The subunits consist of three domains, and a AAAϩ region of homology is localized to the first two domains (9). Many AAAϩ proteins are homohexamers arranged in a symmetric circle (10, 11). The ␥ 3 ␦␦Ј pentamer forms an asymmetric circle, and there is a gap in place of the "missing sixth subunit"...

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Section: The Open Clamp Is a Spiral Lockwashermentioning

confidence: 84%

Replisome Architecture and Dynamics in Escherichia coli

O'Donnell

2006

Journal of Biological Chemistry

126

114

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“…5). A previous study demonstrated that such an open ring conformation exists in the T4 clamp-loading pathway (23). We constructed this model by twisting the intersubunit ␤ sheet formed between subunits and adjusting the dihedral angle of the interdomain loop to keep the distance between W185 and C107 close to 34 Å. Interestingly, with a right-handed opening of the PCNA ring, a gap of Ϸ5 Å can be generated.…”

Section: Discussionmentioning

confidence: 99%

The structure of a ring-opened proliferating cell nuclear antigen–replication factor C complex revealed by fluorescence energy transfer

Zhuang

Yoder

Burgers

et al. 2006

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

Self Cite

104

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We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATP␥S cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.clamp loader ͉ DNA replication T he replisome is a dynamic molecular complex responsible for the duplication of a genomic DNA template at the replication fork. The central components of the replisome are the replicative DNA polymerases that copy both leading-and lagging-strand DNA (reviewed in ref. 1). Processivity of DNA replication is accomplished through a clamp protein that forms a holoenzyme complex with the replicative DNA polymerase (2). The clamp proteins identified in different organisms show a common toroidal architecture with a central channel large enough to encircle duplex DNA (3). Once loaded onto duplex DNA, clamp proteins can freely slide in a 2D space. The eukaryotic clamp proliferating cell nuclear antigen (PCNA) forms a holoenzyme complex with DNA polymerases ␦ and through specific interaction(s) between the polymerase and the PCNA ring (4-6).The task of loading PCNA onto DNA at the primer-template junction is fulfilled by replication factor C (RFC) in an ATP-driven process. Eukaryotic RFC is a heteropentameric protein complex consisting of Rfc1, -2, -3, -4, and -5 subunits. Each Rfc subunit belongs to the AAA ϩ protein family that contains characteristic ATP-binding͞hydrolysis motifs and converts the chemical energy derived from ATP binding and hydrolysis to mechanic force that is required for clamp loading (7). The RFC complex shares partial similarities to its counterpart in lower organisms like bacteriophage T4 and Escherichia coli. However, the small subunits of eukaryotic RFC are more diverged compared with those in prokaryote and archaeon, which hints for a higher complexity in both structural and functional aspects. Indeed, the yeast RFC showed an interesting departure from its prokaryotic counterparts in terms of a step-wise binding of ATP molecules modulated by its interaction with either DNA or PCNA (8). Therefore, a substantial difference is expected in the mechanistic details for clamp loading as compared with its prokaryotic counterpart. Furthermore, additional RFC-like complexes have been identified that share the Rfc2-5 subunits with RFC but carry out specialized functions in the DNA damage checkpoint and in sister chromatid cohesion (reviewed in ref. 9).Although the clamp loaders from the three model systems, including yeast, E. coli, and bacteriophage T4, all use ATP for loading the clamp onto DNA, the timing and amount of ATP binding and of its hydrolysis differ. For the E. coli system, ATP binding is sufficient for opening the ␤ clamp (10), and subseque...

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“…I DA is the donor emission intensity in the presence of the acceptor, and I D is the intensity in the absence of the acceptor. R o is the Forster radius for the donor-acceptor pair used, Trp-CPM, and was set to 29 Å, which has been reported (29,30).…”

Section: Methodsmentioning

confidence: 99%

Modulation of Apolipoprotein A-IV Lipid Binding by an Interaction between the N and C Termini

Tubb

Silva

Tso

et al. 2007

Journal of Biological Chemistry

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Apolipoprotein A-IV (apoA-IV) is a 376-amino acid exchangeable apolipoprotein made in the small intestine of humans. Although it has many proposed roles in vascular disease, satiety, and chylomicron metabolism, there is no known structural basis for these functions. The ability to associate with lipids may be a key step in apoA-IV functionality. We recently identified a single amino acid, Phe 334 , which seems to inhibit the lipid binding capability of apoA-IV. We also found that an intact N terminus was necessary for increased lipid binding of Phe 334 mutants. Here, we identify Trp 12 and Phe 15 as the N-terminal amino acids required for the fast lipid binding seen with the F334A mutant. Furthermore, we found that individual disruption of putative amphipathic ␣-helices 3-11 had little effect on lipid binding, suggesting that the N terminus of apoA-IV may be the operational site for initial lipid binding. We also provide three independent pieces of experimental evidence supporting a direct intramolecular interaction between sequences near amino acids 12/15 and 334. This interaction could represent a unique "switch" mechanism by which apoA-IV changes lipid avidity in vivo.

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Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

Cited by 104 publications

References 62 publications

Replisome Architecture and Dynamics in Escherichia coli

Replisome Architecture and Dynamics in Escherichia coli

The structure of a ring-opened proliferating cell nuclear antigen–replication factor C complex revealed by fluorescence energy transfer

Modulation of Apolipoprotein A-IV Lipid Binding by an Interaction between the N and C Termini

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