Reverse-chaperoning activity of an AAA+ protein

Speed and processivity of replicative DNA polymerases can be enhanced via coupling to a sliding clamp. Due to the closed ring shape of the clamp, a clamp loader protein, belonging to the AAA+ class of ATPases, needs to open the ring-shaped clamp before loading it to DNA. Here, we developed real-time fluorescence assays to study the clamp (PCNA) and the clamp loader (RFC) from the mesophilic archaeon Methanosarcina acetivorans. Unexpectedly, we discovered that RFC can assemble a PCNA ring from monomers in solution. A motion-based DNA polymerization assay showed that the PCNA assembled by RFC is functional. This PCNA assembly activity required the ATP-bound conformation of RFC. Our work demonstrates a reverse-chaperoning activity for an AAA+ protein that can act as a template for the assembly of another protein complex.     

The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases.

The binding of linear and circular forms of DNA to yeast DNA topoisomerase II or its complex with AMPPNP, the nonhydrolyzable beta,gamma-imido analog of ATP, was carried out to probe the ATP analog-induced conformational change of the enzyme. Binding of the ATP analog is shown to convert the enzyme to a circular clamp with an annulet, through which only a linear DNA can pass; subsequent circularization of the bound linear DNA forms a salt-stable catenane between the protein circular clamp and the DNA ring. Analysis of catenane formation between a small DNA ring originally bound to the topoisomerase and a large DNA ring subsequently added, under conditions such that the two do not exchange, supports a model in which a second DNA double-helix can enter the open jaws of a DNA-bound protein clamp, and the closure of the jaws upon ATP-binding traps the second duplex and transports it through an enzyme-operated gate in the first DNA duplex.     

Processivity factor of DNA polymerase and its expanding role in normal and translesion DNA synthesis

Clamp protein or clamp, initially identified as the processivity factor of the replicative DNA polymerase, is indispensable for the timely and faithful replication of DNA genome. Clamp encircles duplex DNA and physically interacts with DNA polymerase. Clamps from different organisms share remarkable similarities in both structure and function. Loading of clamp onto DNA requires the activity of clamp loader. Although all clamp loaders act by converting the chemical energy derived from ATP hydrolysis to mechanical force, intriguing differences exist in the mechanistic details of clamp loading. The structure and function of clamp in normal and translesion DNA synthesis has been subjected to extensive investigations. This review summarizes the current understanding of clamps from three kingdoms of life and the mechanism of loading by their cognate clamp loaders. We also discuss the recent findings on the interactions between clamp and DNA, as well as between clamp and DNA polymerase (both the replicative and specialized DNA polymerases). Lastly the role of clamp in modulating polymerase exchange is discussed in the context of translesion DNA synthesis.     

Distinct roles for ATP binding and hydrolysis at individual subunits of an archaeal clamp loader

Circular clamps are utilised by replicative polymerases to enhance processivity. The topological problem of loading a toroidal clamp onto DNA is overcome by ATP-dependent clamp loader complexes. Different organisms use related protein machines to load clamps, but the mechanisms by which they utilise ATP are surprisingly different. Using mutant clamp loaders that are deficient in either ATP binding or hydrolysis in different subunits, we show how the different subunits of an archaeal clamp loader use ATP binding and hydrolysis in distinct ways at different steps in the loading process. Binding of nucleotide by the large subunit and three of the four small subunits is sufficient for clamp loading. However, ATP hydrolysis by the small subunits is required for release of PCNA to allow formation of the complex between PCNA and the polymerase, while hydrolysis by the large subunit is required for catalytic clamp loading.     

The internal workings of a DNA polymerase clamp-loading machine.

Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader. We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The gamma complex uses ATP to open the beta clamp and assemble it onto DNA. Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp. The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it. The delta' subunit acts as a modulator of the interaction between delta and beta. On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface. The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex.     

Mapping the interaction of DNA with the Escherichia coli DNA polymerase clamp loader complex

Sliding clamps are loaded onto DNA by ATP-dependent clamp loader complexes. A recent crystal structure of a clamp loader-clamp complex suggested an unexpected mechanism for DNA recognition, in which the ATPase subunits of the loader spiral around primed DNA. We report the results of fluorescence-based assays that probe the mechanism of the Escherichia coli clamp loader and show that conserved residues clustered within the inner surface of the modeled clamp loader spiral are critical for DNA recognition, DNA-dependent ATPase activity and clamp release. Duplex DNA with a 5'-overhang single-stranded region (corresponding to correctly primed DNA) stimulates clamp release, as does blunt-ended duplex DNA, whereas duplex DNA with a 3' overhang and single-stranded DNA are ineffective. These results provide evidence for the recognition of DNA within an inner chamber formed by the spiral organization of the ATPase domains of the clamp loader.     

Dynamics of Loading the Escherichia coli DNA Polymerase Processivity Clamp

ABSTRACT

Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein–protein and protein–DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.     

Dynamics of loading the Escherichia coli DNA polymerase processivity clamp

Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein-protein and protein-DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.     

Structure of a sliding clamp on DNA

The structure of the E. coli beta clamp polymerase processivity factor has been solved in complex with primed DNA. Interestingly, the clamp directly binds the DNA duplex and also forms a crystal contact with the ssDNA template strand, which binds into the protein-binding pocket of the clamp. We demonstrate that these clamp-DNA interactions function in clamp loading, perhaps by inducing the ring to close around DNA. Clamp binding to template ssDNA may also serve to hold the clamp at a primed site after loading or during switching of multiple factors on the clamp. Remarkably, the DNA is highly tilted as it passes through the beta ring. The pronounced 22 degrees angle of DNA through beta may enable DNA to switch between multiple factors bound to a single clamp simply by alternating from one protomer of the ring to the other.     

Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation

In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the subsequent binding of polymerase to the loaded clamp. Recently published evidence suggests that holoenzyme assembly in the T4 replisome may take place via more than one pathway [Zhuang, Z., Berdis, A. J., and Benkovic, S. J. (2006) Biochemistry 45, 7976-7989]. To demonstrate unequivocally whether there are multiple pathways leading to the formation of a productive holoenzyme, single-molecule fluorescence microscopy has been used to study the potential clamp loading and holoenzyme assembly pathways on a single-molecule DNA substrate. The results obtained reveal four pathways that foster the formation of a functional holoenzyme on DNA: (1) clamp loader-clamp complex binding to DNA followed by polymerase, (2) clamp loader binding to DNA followed by clamp and then polymerase, (3) clamp binding to DNA followed by clamp loader and then polymerase, and (4) polymerase binding to DNA followed by the clamp loader-clamp complex. In all cases, MgATP is required. The possible physiological significance of the various assembly pathways is discussed in the context of replication initiation and lagging strand synthesis during various stages of T4 phage replication.     

Protein trafficking on sliding clamps

The sliding clamps of chromosomal replicases are acted upon by both the clamp loader and DNA polymerase. Several other proteins and polymerases also interact with the clamp. These proteins bind the clamp at the same spot and use it in sequential fashion. First the clamp loader must bind the clamp in order to load it onto DNA, but directly thereafter the clamp loader must clear away from the clamp so it can be used by the replicative DNA polymerase. At the end of replication, the replicase is ejected from the clamp, which presumably allows the clamp to interact with yet other proteins after its use by the replicase. This paper describes how different proteins in the Escherichia coli replicase, DNA polymerase III holoenzyme, coordinate their traffic flow on the clamp. The mechanism by which traffic flow on the beta clamp is directed is based on competition of the proteins for the clamp, where DNA structure modulates the competition. It seems likely that the principles will generalize to a traffic flow of other factors on these circular clamp proteins.     

The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.     

The Escherichia coli clamp loader can actively pry open the β-sliding clamp

Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.     

An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex

In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form in solution raises the possibility of other pathways for clamp loading. In this study, an alternative clamp loading sequence is evaluated in which gp44/62 first recognizes the DNA substrate and then sequesters the clamp from solution and loads it onto DNA. This pathway differs in terms of the initial formation of a gp44/62-DNA complex that is capable of loading gp45. In this work, we demonstrate ATP-dependent DNA binding by gp44/62. Among various DNA structures that were tested, gp44/62 binds specifically to primer-template DNA but not to single-stranded DNA or blunt-end duplex DNA. By tracing the dynamic clamp closing with pre-steady-state FRET measurements, we show that the clamp loader-DNA complex is functional in clamp loading. Furthermore, pre-steady-state ATP hydrolysis experiments suggest that 1 equiv of ATP is hydrolyzed when gp44/62 binds to DNA, and additional ATP hydrolysis is associated with the completion of the clamp loading process. We also investigated the detailed kinetics of binding of MANT-nucleotide to gp44/62 through stopped-flow FRET and demonstrated a conformational change as the result of ATP, but not ADP binding. The collective kinetic data allowed us to propose and evaluate a sequence of steps describing this alternative pathway for clamp loading and holoenzyme formation.     

Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus

The proliferating cell nuclear antigen (PCNA) is now recognized as one of the key proteins in DNA metabolic events because of its direct interactions with many proteins involved in important cellular processes. We have determined the crystal structure of PCNA from a hyperthermophilic archaeon, Pyrococcus furiosus (pfuPCNA), at 2.1 A resolution. pfuPCNA forms a toroidal, ring-shaped structure consisting of homotrimeric molecules, which is also observed in the PCNA crystals from human and yeast. The overall structure of pfuPCNA is highly conserved with other PCNA proteins, as well as with the bacterial ss clamp and the bacteriophage gp45. This result shows that the three-dimensional structure of the sliding clamp is conserved in the three domains of life. pfuPCNA has two remarkable features compared with the human and yeast PCNA molecules: it has more ion pairs and fewer intermolecular main chain hydrogen bonds. The former may contribute to the thermal stability of pfuPCNA, and the latter may be the cause of the stimulatory effect of pfuPCNA on the DNA synthesizing activity of P. furiosus DNA polymerases in the absence of the clamp loader replication factor C in vitro.     

Examination of the role of the clamp-loader and ATP hydrolysis in the formation of the bacteriophage T4 polymerase holoenzyme

Transient kinetic analyses further support the role of the clamp-loader in bacteriophage T4 as a catalyst which loads the clamp onto DNA through the sequential hydrolysis of two molecules of ATP before and after addition of DNA. Additional rapid-quench and pulse-chase experiments have documented this stoichiometry. The events of ATP hydrolysis have been related to the opening/closing of the clamp protein through fluorescence resonance energy transfer (FRET). In the absence of a hydrolysable form of ATP, the distance across the subunit interface of the clamp does not increase as measured by intramolecular FRET, suggesting gp45 cannot be loaded onto DNA. Therefore, ATP hydrolysis by the clamp-loader appears to open the clamp wide enough to encircle DNA easily. Two additional molecules of ATP then are hydrolyzed to close the clamp onto DNA. The presence of an intermolecular FRET signal indicated that the dissociation of the clamp-loader from this complex occurred after guiding the polymerase onto the correct face of the clamp bound to DNA. The final holoenzyme complex consists of the clamp, DNA, and the polymerase. Although this sequential assembly mechanism can be generally applied to most other replication systems studied to date, the specifics of ATP utilization seem to vary across replication systems.     

Computational analysis of DNA replicases in double-stranded DNA viruses: relationship with the genome size

Genome duplication in free-living cellular organisms is performed by DNA replicases that always include a DNA polymerase, a DNA sliding clamp and a clamp loader. What are the evolutionary solutions for DNA replicases associated with smaller genomes? Are there some general principles? To address these questions we analyzed DNA replicases of double-stranded (ds) DNA viruses. In the process we discovered highly divergent B-family DNA polymerases in phiKZ-like phages and remote sliding clamp homologs in Ascoviridae family and Ma-LMM01 phage. The analysis revealed a clear dependency between DNA replicase components and the viral genome size. As the genome size increases, viruses universally encode their own DNA polymerases and frequently have homologs of DNA sliding clamps, which sometimes are accompanied by clamp loader subunits. This pattern is highly non-random. The absence of sliding clamps in large viral genomes usually coincides with the presence of atypical polymerases. Meanwhile, sliding clamp homologs, not accompanied by clamp loaders, have an elevated positive electrostatic potential, characteristic of non-ring viral processivity factors that bind the DNA directly. Unexpectedly, we found that similar electrostatic properties are shared by the eukaryotic 9-1-1 clamp subunits, Hus1 and, to a lesser extent, Rad9, also suggesting the possibility of direct DNA binding.     

Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III beta subunit or beta clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.     

An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.