Regulation of Interactions with Sliding Clamps During DNA Replication and Repair

The molecular machines that replicate the genome consist of many interacting components. Essential to the organization of the replication machinery are ring-shaped proteins, like PCNA (Proliferating Cell Nuclear Antigen) or the β- clamp, collectively named sliding clamps. They encircle the DNA molecule and slide on it freely and bidirectionally. Sliding clamps are typically associated to DNA polymerases and provide these enzymes with the processivity required to synthesize large chromosomes. Additionally, they interact with a large array of proteins that perform enzymatic reactions on DNA, targeting and orchestrating their functions. In recent years there have been a large number of studies that have analyzed the structural details of how sliding clamps interact with their ligands. However, much remains to be learned in relation to how these interactions are regulated to occur coordinately and sequentially. Since sliding clamps participate in reactions in which many different enzymes bind and then release from the clamp in an orchestrated way, it is critical to analyze how these changes in affinity take place. In this review I focus the attention on the mechanisms by which various types of enzymes interact with sliding clamps and what is known about the regulation of this binding. Especially I describe emerging paradigms on how enzymes switch places on sliding clamps during DNA replication and repair of prokaryotic and eukaryotic genomes.     

Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation

The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.     

The ring-type polymerase sliding clamp family

Ring-type polymerases consist of a DNA polymerase, a ring-shaped sliding clamp protein and a clamp-loading complex. Sliding clamp proteins are found in all organisms and are called proliferating cell nuclear antigen (PCNA) in eukaryotes and the β clamp in prokaryotes. Both PCNA and β form a ring around DNA, which is made up of two subunits of three domains each in β but three subunits of two domains each in PCNA. Despite this difference and a lack of detectable sequence homology, the structures of the two rings are very similar. The sliding clamp slides along DNA and tethers the polymerase to the DNA, enabling rapid and processive DNA replication.     

A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA

DNA sliding clamps encircle DNA and provide binding sites for many DNA-processing enzymes. However, it is largely unknown how sliding clamps like proliferating cell nuclear antigen (PCNA) coordinate multistep DNA transactions. We have determined structures of Sulfolobus solfataricus DNA ligase and heterotrimeric PCNA separately by X-ray diffraction and in complex by small-angle X-ray scattering (SAXS). Three distinct PCNA subunits assemble into a protein ring resembling the homotrimeric PCNA of humans but with three unique protein-binding sites. In the absence of nicked DNA, the Sulfolobus solfataricus DNA ligase has an open, extended conformation. When complexed with heterotrimeric PCNA, the DNA ligase binds to the PCNA3 subunit and ligase retains an open, extended conformation. A closed, ring-shaped conformation of ligase catalyzes a DNA end-joining reaction that is strongly stimulated by PCNA. This open-to-closed switch in the conformation of DNA ligase is accommodated by a malleable interface with PCNA that serves as an efficient platform for DNA ligation.     

The cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer

The human cytomegalovirus DNA polymerase consists of a catalytic subunit, UL54, and a presumed processivity factor, UL44. We have solved the crystal structure of residues 1-290 of UL44 to 1.85 A resolution by multiwavelength anomalous dispersion. The structure reveals a dimer of UL44 in the shape of a C clamp. Each monomer of UL44 shares its overall fold with other processivity factors, including herpes simplex virus UL42, which is a monomer that binds DNA directly, and the sliding clamp, PCNA, which is a trimer that surrounds DNA, although these proteins share no obvious sequence homology. Analytical ultracentrifugation and gel filtration measurements demonstrated that UL44 also forms a dimer in solution, and substitution of large hydrophobic residues along the homodimer interface with alanine disrupted dimerization and decreased DNA binding. UL44 represents a hybrid processivity factor as it binds DNA directly like UL42, but forms a C clamp that may surround DNA like PCNA.     

Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA

DNA polymerases are co-ordinated by sliding clamps (PCNA/β-clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full-length Y-family polymerase, Dpo4, in complex with heterodimeric PCNA1–PCNA2 at 2.05 Å resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo- or DNA-bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C-terminal peptide of Dpo4 becomes structured with a 3₁₀ helix and dominates the specific binding. The Y-family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation-dependent protein–protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding-coupled multiple conformations of a Y-family polymerase facilitate its recruitment and co-ordination on the sliding clamp. The conformational flexibility would turn the error-prone Y-family polymerase off when more efficient high-fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.     

Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure

Chromosomal replicases of cellular organisms utilize a ring shaped protein that encircles DNA as a mobile tether for high processivity in DNA synthesis. These "sliding clamps" have sufficiently large linear diameters to encircle duplex DNA and are perhaps even large enough to slide over certain DNA secondary structural elements. This report examines the Escherichia coli beta and human proliferating cell nuclear antigen clamps for their ability to slide over various DNA secondary structures. The results show that these clamps are capable of traversing a 13-nucleotide ssDNA loop, a 4-base pair stem-loop, a 4-nucleotide 5' tail, and a 15-mer bubble within the duplex. However, upon increasing the size of these structures (20-nucleotide loop, 12-base pair stem-loop, 28-nucleotide 5' tail, and 20-nucleotide bubble) the sliding motion of the beta and proliferating cell nuclear antigen over these elements is halted. Studies of the E. coli replicase, DNA polymerase III holoenzyme, in chain elongation with the beta clamp demonstrate that upon encounter with an oligonucleotide annealed in its path, it traverses the duplex and resumes synthesis on the 3' terminus of the oligonucleotide. This sliding and resumption of synthesis occurs even when the oligonucleotide contains a secondary structure element, provided the beta clamp can traverse the structure. However, upon encounter with a downstream oligonucleotide containing a large internal secondary structure, the holoenzyme clears the obstacle by strand displacing the oligonucleotide from the template. Implications of these protein dynamics to DNA transactions are discussed.     

High resolution structure of an oligomeric eye lens beta-crystallin. Loops, arches, linkers and interfaces in beta B2 dimer compared to a monomeric gamma-crystallin.

beta-Crystallins are polydisperse, oligomeric structural proteins that have a major role in forming the high refractive index of the eye lens. Using single crystal X-ray crystallography with molecular replacement, the structure of beta B2 dimer has been solved at 2.1 A resolution. Each subunit comprises an N and C-terminal domain that are very similar and each domain is formed from two similar "Greek key" motifs related by a local dyad. Sequence differences in the internally quadruplicated molecules, analysed in terms of their beta-sheets, hairpins and arches, give rise to structural differences in the motifs. Whereas the related family of gamma-crystallins are monomers, beta-crystallins are always oligomers. In the beta B2 subunit, the domains, each comprising two motifs, are separated by an extended linking peptide. A crystallographic 2-fold axis relates the two subunits of the dimer so that the N-terminal domain of one subunit of beta B2 and the C-terminal domain of the symmetry-related subunit are topologically equivalent to the two covalently connected domains of gamma B-crystallin. The intersubunit domain interface is very similar to the intradomain interface of gamma B, although many sequence differences have resulted in an increase in polar interactions between domains in beta B2. Comparison of the structures of beta B2 and gamma B-crystallins shows that the two families differ largely in the conformation of their connecting peptides. A further extensive lattice contact indicates a tetramer with 222 symmetry. The ways in which insertions and extensions in the beta-crystallin effect oligomer interactions are described. The two kinds of crystallin are analysed for structural features that account for their different stabilities. These studies are a basis for understanding formation of higher aggregates in the lens.     

Interplay of clamp loader subunits in opening the beta sliding clamp of Escherichia coli DNA polymerase III holoenzyme

The Escherichia coli beta dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA. The gamma complex (gammadeltadelta'chipsi) clamp loader couples ATP to the opening and closing of beta in assembly of the ring onto DNA. These proteins are functionally and structurally conserved in all cells. The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp. The delta subunit of the E. coli gamma complex clamp loader is known to bind beta and open it by parting one of the dimer interfaces. This study demonstrates that other subunits of gamma complex also bind beta, although weaker than delta. The gamma subunit like delta, affects the opening of beta, but with a lower efficiency than delta. The delta' subunit regulates both gamma and delta ring opening activities in a fashion that is modulated by ATP interaction with gamma. The implications of these actions for the workings of the E. coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed.     

Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp.

The crystal structure of the beta subunit (processivity factor) of DNA polymerase III holoenzyme has been determined at 2.5 A resolution. A dimer of the beta subunit (M(r) = 2 x 40.6 kd, 2 x 366 amino acid residues) forms a ring-shaped structure lined by 12 alpha helices that can encircle duplex DNA. The structure is highly symmetrical, with each monomer containing three domains of identical topology. The charge distribution and orientation of the helices indicate that the molecule functions by forming a tight clamp that can slide on DNA, as shown biochemically. A potential structural relationship is suggested between the beta subunit and proliferating cell nuclear antigen (PCNA, the eukaryotic polymerase delta [and epsilon] processivity factor), and the gene 45 protein of the bacteriophage T4 DNA polymerase.     

Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC

Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.     

Sliding clamp of the bacteriophage T4 polymerase has open and closed subunit interfaces in solution.

The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (beta clamp), and yeast (PCNA) are required for processive DNA synthesis by their cognate DNA polymerases. The X-ray crystal structures of all three of these clamps have been shown to be closed, circular complexes. This paper reports investigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation, fluorescence, and hydrodynamic modeling. Mutants of gp45 with inter- and intrasubunit disulfide bonds were created to alter the solution structure of gp45, with additional mutagenesis used to investigate the importance of the proline-rich loop region found between the two domains of each gp45 monomer. The wild-type gp45 trimer assembles from monomers cooperatively with a dissociation constant of 0.21 microM2 and values between 0.088 and 0. 32 microM2 for the mutants. Velocity ultracentrifugation experiments showed that wild-type gp45 possesses a sedimentation coefficient strongly dependent on concentration, typical of asymmetric or elongated molecules, that when extrapolated to zero concentration yields a sedimentation coefficient of 4.0 S. The loop and the disulfide mutants exhibited sedimentation coefficients with little concentration dependence, typical of symmetric or spherical molecules, that when extrapolated to zero concentration yielded sedimentation coefficients of 4.4-4.8 S. The lower sedimentation coefficient in the former case is consistent with wild-type gp45 being more asymmetric or elongated than the mutant forms. Fluorescence-resonance energy-transfer experiments were used to measure the distance between two amino acids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interface. For an intrasubunit disulfide mutant, the distance between these two amino acids was determined to be 19 A (14 A in the X-ray crystal structure), consistent with a closed complex. For the mutants without intrasubunit disulfides, the efficiency of fluorescence-resonance energy transfer was in accord with a model of gp45 being an open complex composed of two closed subunit interfaces and a third open interface separated by a distance of 35-38 A. The collective data supplemented with hydrodynamic modeling were consistent with gp45 subunit separation achieved within the plane of the gp45 ring.     

Linear diffusion on DNA despite high-affinity binding by a DNA polymerase processivity factor.

The oligomeric "sliding clamp" processivity factors, such as PCNA, are thought to rely on a loose, topological association with DNA to slide freely along dsDNA. Unlike PCNA, the processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer and has an intrinsic affinity for dsDNA that is remarkably high for a sequence-independent DNA binding protein. Using a DNase footprinting assay, we demonstrate that UL42 translocates with the catalytic subunit of the polymerase during chain elongation. In addition, footprinting and electrophoretic mobility shift assays show that, despite its tight DNA binding, UL42 is capable of linear diffusion on DNA at a rate of between 17 and 47 bp/s. Our results thus suggest that, despite profound biochemical differences with the sliding clamps, UL42 can freely slide downstream with the catalytic subunit during DNA replication.     

The Mechanical Properties of PCNA: Implications for the Loading and Function of a DNA Sliding Clamp

Sliding clamps are toroidal proteins that encircle DNA and act as mobile platforms for DNA replication and repair machinery. To be loaded onto DNA, the eukaryotic sliding clamp Proliferating Cell Nuclear Antigen (PCNA) must be splayed open at one of the subunit-subunit interfaces by the ATP-dependent clamp loader, Replication Factor C, whose clamp-interacting sites form a right-handed spiral. Earlier molecular dynamics (MD) studies suggested that when PCNA opens, it preferentially adopts a right-handed spiral to match the spiral of the clamp loader. Here, analysis of considerably longer MD simulations shows that although the opened form of PCNA can achieve conformations matching the helical pitch of Replication Factor C, it is not biased toward a right-handed spiral structure. A coarse-grained elastic model was also built; its strong correspondence to the all-atom MD simulations of PCNA suggests that the behavior of the open clamp is primarily due to elastic deformation governed by the topology of the clamp domains. The elastic model was further used to construct the energy landscape of the opened PCNA clamp, including conformations that would allow binding to the clamp loader and loading onto double-stranded DNA. A picture of PCNA emerges of a rather flexible protein that, once opened, is mechanically compliant in the clamp opening process.     

A novel heterotetrameric structure of the crenarchaeal PCNA2-PCNA3 complex

Proliferating cell nuclear antigen (PCNA) is a key protein that orchestrates the arrangement of DNA-processing proteins on DNA during DNA metabolism. In crenarchaea, PCNA forms a heterotrimer (PCNA123) consisting of PCNA1, PCNA2, and PCNA3, while in most eukaryotes and many archaea PCNAs form a homotrimer. Interestingly, unique oligomeric PCNAs from Sulfolobus tokodaii were reported in which PCNA2 and PCNA3 form a heterotrimer without PCNA1. In this paper, we describe the crystal structure of the stoPCNA2-stoPCNA3 complex. While most DNA sliding clamps form ring-shaped structures, our crystal structure showed an elliptic ring-like heterotetrameric complex, differing from a previous reports. Furthermore, we investigated the composition and the dimension of the stoPCNA2-stoPCNA3 complex in the solution using gel-filtration column chromatography and small-angle X-ray scattering analyses, respectively. These results indicate that stoPCNA2 and stoPCNA3 form the heterotetramer in solution. Based on our heterotetrameric structure, we propose a possible biological role for the heterotetrameric complex as a Holliday junction clamp.     

Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage

The protein encoded by gene 45 of T4 bacteriophage (gene 45 protein or gp45), is responsible for tethering the catalytic subunit of T4 DNA Polymerase to DNA during high-speed replication. Also referred to as a sliding DNA clamp, gp45 is similar in its function to the processivity factors of bacterial and eukaryotic DNA polymerases, the beta-clamp and PCNA, respectively. Crystallographic analysis has shown that the beta-clamp and PCNA form highly symmetrical ring-shaped structures through which duplex DNA can be threaded. Gp45 shares no sequence similarity with beta-clamp or PCNA, and sequence comparisons have not been able to establish whether it adopts a similar structure. We have determined the crystal structure of gp45 from T4 bacteriophage at 2.4 A resolution, using multiple isomorphous replacement. The protein forms a trimeric ring-shaped assembly with overall dimensions that are similar to those of the bacterial and eukaryotic processivity factors. Each monomer of gp45 contains two domains that are very similar in chain fold to those of beta-clamp and PCNA. Despite an overall negative charge, the inner surface of the ring is in a region of positive electrostatic potential, consistent with a mechanism in which DNA is threaded through the ring.     

Molecular mechanism of domain swapping in proteins: an analysis of slower motions

Domain swapping is a structural phenomenon that plays an important role in the mechanism of oligomerization of some proteins. The monomer units in the oligomeric structure become entangled with each other. Here we investigate the mechanism of domain swapping in diphtheria toxin and the structural criteria required for it to occur by analyzing the slower modes of motion with elastic network models, Gaussian network model and anisotropic network model. We take diphtheria toxin as a representative of this class of domain-swapped proteins and show that the domain, which is being swapped in the dimeric state, rotates and twists, in going from the "open" to the "closed" state, about a hinge axis that passes through the middle of the loop extending between two domains. A combination of the intra- and intermolecular contacts of the dimer is almost equivalent to that of the monomer, which shows that the relative orientations of the residues in both forms are almost identical. This is also reflected in the calculated B-factors when compared with the experimentally determined B-factors in x-ray crystal structures. The slowest modes of both the monomer and dimer show a common hinge centered on residue 387. The differences in distances between the monomer and the dimer also shows the hinge at nearly the same location (residue 381). Finally, the first three dominant modes of anisotropic network model together shows a twisting motion about the hinge centered on residue 387. We further identify the location of hinges for a set of another 12 domain swapped proteins and give the quantitative measures of the motions of the swapped domains toward their "closed" state, i.e., the overlap and correlation between vectors.     

Replication Clamps and Clamp Loaders

To achieve the high degree of processivity required for DNA replication, DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for replication. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process.During each round of DNA replication, thousands to billions of nucleotides must be faithfully copied in a short period of time. However, by themselves, replicative DNA polymerases are distributive, synthesizing only ten or so nucleotides of complementary DNA before dissociating. To achieve the high degree of processivity required for efficient DNA replication, replicative DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. Such an association effectively tethers the polymerase to DNA, substantially increasing the amount of continuous replication. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for interaction with DNA polymerases. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process.     

Solution structure of the disulfide-linked dimer of human intestinal trefoil factor (TFF3): the intermolecular orientation and interactions are markedly different from those of other dimeric trefoil proteins

The trefoil protein TFF3 forms a homodimer (via a disulfide linkage) that is thought to have increased biological activity over the monomer. The solution structure of the TFF3 dimer has been determined by NMR and compared with the structure of the TFF3 monomer and with other trefoil dimer structures (TFF1 and TFF2). The most significant structural differences between the trefoil domain in the monomer and dimer TFF3 are in the orientations of the N-terminal 3(10)-helix (residues 10-12) and in the presence in the dimer of an additional 3(10)-helix (residues 53-55) outside of the core region. The TFF3 dimer forms a more compact structure as compared with the TFF1 dimer where the two trefoil domains are connected by a flexible region with the monomer units being at variable distances from each other and in many different orientations. Although TFF2 is also a compact structure, the dispositions of its monomer units are very different from those of TFF3. The structural differences between the dimers result in the two putative receptor/ligand binding sites that remain solvent exposed in the dimeric structures having very different dispositions in the different dimers. Such differences have significant implications for the mechanism of action and functional specificity for the TFF class of proteins.     

Replication factor C is a more effective proliferating cell nuclear antigen (PCNA) opener than the checkpoint clamp loader, Rad24-RFC

Clamp loaders from all domains of life load clamps onto DNA. The clamp tethers DNA polymerases to DNA to increase the processivity of synthesis as well as the efficiency of replication. Here, we investigated proliferating cell nuclear antigen (PCNA) binding and opening by the Saccharomyces cerevisiae clamp loader, replication factor C (RFC), and the DNA damage checkpoint clamp loader, Rad24-RFC, using two separate fluorescence intensity-based assays. Analysis of PCNA opening by RFC revealed a two-step reaction in which RFC binds PCNA before opening PCNA rather than capturing clamps that have transiently and spontaneously opened in solution. The affinity of RFC for PCNA is about an order of magnitude lower in the absence of ATP than in its presence. The affinity of Rad24-RFC for PCNA in the presence of ATP is about an order magnitude weaker than that of RFC for PCNA, similar to the RFC-PCNA interaction in the absence of ATP. Importantly, fewer open clamp loader-clamp complexes are formed when PCNA is bound by Rad24-RFC than when bound by RFC.