Crystal structure of the herpes simplex virus 1 DNA polymerase

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 A resolution. The structural similarity of this polymerase to other alpha polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.     

Crystal structure of a DinB family error-prone DNA polymerase from Sulfolobus solfataricus.

A new group of error-prone DNA polymerases overcomes the blockage posed to normal DNA replication by damaged template bases, suggesting an active site with a loose, flexible pocket that accommodates aberrant DNA structures. We have determined a 2.8 A resolution crystal structure of the Sulfolobus solfataricus Dbh protein, a DNA translesion polymerase closely related to Escherichia coli DNA polymerase IV and human polymerase kappa. A high error rate is observed for the Dbh polymerase in a range of 10(-2)-10(-3) for all 12 base substitution mispairs. The crystal structure of Dbh reveals an overall architecture resembling other DNA polymerases but has unique features that are likely to contribute to error-prone synthesis, including -1 frameshifting mutations.     

Crystal structure of the catalytic core of human DNA polymerase kappa

We present the crystal structure of the catalytic core of human DNA polymerase kappa (hPolkappa), the first structure of a human Y-family polymerase. hPolkappa is implicated in the proficient extension of mispaired primer termini on undamaged DNAs, and in the extension step of lesion bypass. The structure reveals a stubby "fingers" subdomain, which despite its small size appears to be tightly restrained with respect to a putative templating base. The structure also reveals a novel "thumb" subdomain that provides a basis for the importance of the N-terminal extension unique to hPolkappa. And, most surprisingly, the structure reveals the polymerase-associated domain (PAD) juxtaposed on the dorsal side of the "palm" subdomain, as opposed to the fingers subdomain. Together, these properties suggest that the hPolkappa active site is constrained at the site of the templating base and incoming nucleotide, but the polymerase is less constrained following translocation of the lesion.     

Crystal structure of a DinB lesion bypass DNA polymerase catalytic fragment reveals a classic polymerase catalytic domain.

The UmuC/DinB family of bypass polymerases is responsible for translesion DNA synthesis and includes the human polymerases eta, iota, and kappa. We determined the 2.3 A resolution crystal structure of a catalytic fragment of the DinB homolog (Dbh) polymerase from Sulfolobus solfataricus and show that it is nonprocessive and can bypass an abasic site. The structure of the catalytic domain is nearly identical to those of most other polymerase families. Homology modeling suggests that there is minimal contact between protein and DNA, that the nascent base pair binding pocket is quite accessible, and that the enzyme is already in a closed conformation characteristic of ternary polymerase complexes. These observations afford insights into the sources of low fidelity and low processivity of the UmuC/DinB polymerases.     

Crystal structure of a DNA-dependent RNA polymerase (DNA primase)

Primases are essential components of the DNA replication apparatus in every organism. They catalyze the synthesis of oligoribonucleotides on single-stranded DNA, which subsequently serve as primers for the replicative DNA polymerases. In contrast to bacterial primases, the archaeal enzymes are closely related to their eukaryotic counterparts. We have solved the crystal structure of the catalytic primase subunit from the hyperthermophilic archaeon Pyrococcus furiosus at 2.3 A resolution by multiwavelength anomalous dispersion methods. The structure shows a two-domain arrangement with a novel zinc knuckle motif located in the primase (prim) domain. In this first structure of a complete protein of the archaeal/eukaryotic primase family, the arrangement of the catalytically active residues resembles the active sites of various DNA polymerases that are unrelated in fold.     

DNA structure and polymerase fidelity.

The accuracy of DNA replication results from both the intrinsic DNA polymerase fidelity and the DNA sequence. Although the recent structural studies on polymerases have brought new insights on polymerase fidelity, the role of DNA sequence and structure is less well understood. Here, the analysis of the crystal structures of hotspots for polymerase slippage including (CA)n and (A)n tracts in different intermolecular contexts reveals that, in the B-form, these sequences share common structural alterations which may explain the high rate of replication errors. In particular, a two-faced "Janus-like" structure with shifted base-pairs in the major groove but an apparent normal geometry in the minor groove constitutes a molecular decoy specifically suitable to mislead the polymerases. A model of the rat polymerase beta bound to this structure suggests that an altered conformation of the nascent template-primer duplex can interfere with correct nucleotide incorporation by affecting the geometry of the active site and breaking the rules of base-pairing, while at the same time escaping enzymatic mechanisms of error discrimination which scan for the correct geometry of the minor groove.In contrast, by showing that the A-form greatly attenuates the sequence-dependent structural alterations in hotspots, this study suggests that the A-conformation of the nascent template-primer duplex at the vicinity of the polymerase active site will contribute to fidelity. The A-form may play the role of a structural buffer which preserves the correct geometry of the active site for all sequences. The detailed comparison of the conformation of the nascent template-primer duplex in the available crystal structures of DNA polymerase-DNA complexes shows that polymerase beta, the least accurate enzyme, is unique in binding to a B-DNA duplex even close to its active site. This model leads to several predictions which are discussed in the light of published experimental data.     

Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III

Bacterial replicative DNA polymerases such as Polymerase III (Pol III) share no sequence similarity with other polymerases. The crystal structure, determined at 2.3 A resolution, of a large fragment of Pol III (residues 1-917), reveals a unique chain fold with localized similarity in the catalytic domain to DNA polymerase beta and related nucleotidyltransferases. The structure of Pol III is strikingly different from those of members of the canonical DNA polymerase families, which include eukaryotic replicative polymerases, suggesting that the DNA replication machinery in bacteria arose independently. A structural element near the active site in Pol III that is not present in nucleotidyltransferases but which resembles an element at the active sites of some canonical DNA polymerases suggests that, at a more distant level, all DNA polymerases may share a common ancestor. The structure also suggests a model for interaction of Pol III with the sliding clamp and DNA.     

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.     

Crystal structure of an antigen-binding fragment bound to single-stranded DNA.

Antibodies to DNA are characteristic of the autoimmune disease systemic lupus erythematosus (SLE) and they also serve as models for the study of protein-DNA recognition. Anti-DNA antibodies often play an important role in disease pathogenesis by mediating kidney damage via antibody-DNA immune complex formation. The structural underpinnings of anti-DNA antibody pathogenicity and antibody-DNA recognition, however, are not well understood, due in part to the lack of direct, experimental three-dimensional structural information on antibody-DNA complexes. To address these issues for anti-single-stranded DNA antibodies, we have determined the 2.1 A crystal structure of a recombinant Fab (DNA-1) in complex with dT5. DNA-1 was previously isolated from a bacteriophage Fab display library from the immunoglobulin repertoire of an SLE-prone mouse. The structure shows that DNA-1 binds oligo(dT) primarily by sandwiching thymine bases between Tyr side-chains, which allows the bases to make sequence-specific hydrogen bonds. The critical stacking Tyr residues are L32, L49, H100, and H100A, while His L91 and Asn L50 contribute hydrogen bonds. Comparison of the DNA-1 structure to other anti-nucleic acid Fab structures reveals a common ssDNA recognition module consisting of Tyr L32, a hydrogen bonding residue at position L91, and an aromatic side-chain from the tip of complementarity determining region H3. The structure also provides a framework for interpreting previously determined thermodynamics data, and this analysis suggests that hydrophobic desolvation might underlie the observed negative enthalpy of binding. Finally, Arg side-chains from complementarity determining region H3 appear to play a novel role in DNA-1. Rather than forming ion pairs with dT5, Arg contributes to oligo(dT) recognition by helping to maintain the structural integrity of the combining site. This result is significant because antibody pathogenicity is thought to be correlated to the Arg content of anti-DNA antibody hypervariable loops.     

Crystal structure of a DNA polymerase sliding clamp from a Gram-positive bacterium

Background  

Sliding DNA clamps are processivity factors that are required for efficient DNA replication. DNA polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle DNA and thereby link the polymerase enzyme to the DNA substrate. Although the structures of sliding clamps from Gram-negative bacteria (E. coli), eukaryotes, archaea, and T4-like bacteriophages are well-known, the structure of a sliding clamp from Gram-positive bacteria has not been reported previously.     

Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The crystal structure of family B DNA polymerase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) was determined. KOD DNA polymerase exhibits the highest known extension rate, processivity and fidelity. We carried out the structural analysis of KOD DNA polymerase in order to clarify the mechanisms of those enzymatic features. Structural comparison of DNA polymerases from hyperthermophilic archaea highlighted the conformational difference in Thumb domains. The Thumb domain of KOD DNA polymerase shows an "opened" conformation. The fingers subdomain possessed many basic residues at the side of the polymerase active site. The residues are considered to be accessible to the incoming dNTP by electrostatic interaction. A beta-hairpin motif (residues 242-249) extends from the Exonuclease (Exo) domain as seen in the editing complex of the RB69 DNA polymerase from bacteriophage RB69. Many arginine residues are located at the forked-point (the junction of the template-binding and editing clefts) of KOD DNA polymerase, suggesting that the basic environment is suitable for partitioning of the primer and template DNA duplex and for stabilizing the partially melted DNA structure in the high-temperature environments. The stabilization of the melted DNA structure at the forked-point may be correlated with the high PCR performance of KOD DNA polymerase, which is due to low error rate, high elongation rate and processivity.     

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3',5'-phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA-protein world.     

Crystal structure of Pfu, the high fidelity DNA polymerase from Pyrococcus furiosus

We have determined a 2.6 Å resolution crystal structure of Pfu DNA polymerase, the most commonly used high fidelity PCR enzyme, from Pyrococcus furiosus. Although the structures of Pfu and KOD1 are highly similar, the structure of Pfu elucidates the electron density of the interface between the exonuclease and thumb domains, which has not been previously observed in the KOD1 structure. The interaction of these two domains is known to coordinate the proofreading and polymerization activity of DNA polymerases, especially via H147 that is present within the loop (residues 144–158) of the exonuclease domain. In our structure of Pfu, however, E148 rather than H147 is located at better position to interact with the thumb domain. In addition, the structural analysis of Pfu and KOD1 shows that both the Y-GG/A and β-hairpin motifs of Pfu are found to differ with that of KOD1, and may explain differences in processivity. This information enables us to better understand the mechanisms of polymerization and proofreading of DNA polymerases.     

Crystal structure of the chi:psi sub-assembly of the Escherichia coli DNA polymerase clamp-loader complex.

The chi (chi) and psi (psi) subunits of Escherichia coli DNA polymerase III form a heterodimer that is associated with the ATP-dependent clamp-loader machinery. In E. coli, the chi:psi heterodimer serves as a bridge between the clamp-loader complex and the single-stranded DNA-binding protein. We determined the crystal structure of the chi:psi heterodimer at 2.1 A resolution. Although neither chi (147 residues) nor psi (137 residues) bind to nucleotides, the fold of each protein is similar to the folds of mononucleotide-(chi) or dinucleotide-(psi) binding proteins, without marked similarity to the structures of the clamp-loader subunits. Genes encoding chi and psi proteins are found to be readily identifiable in several bacterial genomes and sequence alignments showed that residues at the chi:psi interface are highly conserved in both proteins, suggesting that the heterodimeric interaction is of functional significance. The conservation of surface-exposed residues is restricted to the interfacial region and to just two other regions in the chi:psi complex. One of the conserved regions was found to be located on chi, distal to the psi interaction region, and we identified this as the binding site for a C-terminal segment of the single-stranded DNA-binding protein. The other region of sequence conservation is localized to an N-terminal segment of psi (26 residues) that is disordered in the crystal structure. We speculate that psi is linked to the clamp-loader complex by this flexible, but conserved, N-terminal segment, and that the chi:psi unit is linked to the single-stranded DNA-binding protein via the distal surface of chi. The base of the clamp-loader complex has an open C-shaped structure, and the shape of the chi:psi complex is suggestive of a loose docking within the crevice formed by the open faces of the delta and delta' subunits of the clamp-loader.     

Crystal structure of a DNA Holliday junction.

DNA recombination is a universal biological event responsible both for the generation of genetic diversity and for the maintenance of genome integrity. A four-way DNA junction, also termed Holliday junction, is the key intermediate in nearly all recombination processes. This junction is the substrate of recombination enzymes that promote branch migration or catalyze its resolution. We have determined the crystal structure of a four-way DNA junction by multiwavelength anomalous diffraction, and refined it to 2.16 A resolution. The structure has two-fold symmetry, with pairwise stacking of the double-helical arms, which form two continuous B-DNA helices that run antiparallel, cross in a right-handed way, and contain two G-A mismatches. The exchanging backbones form a compact structure with strong van der Waals contacts and hydrogen bonds, implying that a conformational change must occur for the junction to branch-migrate or isomerize. At the branch point, two phosphate groups from one helix occupy the major groove of the other one, establishing sequence-specific hydrogen bonds. These interactions, together with different stacking energies and steric hindrances, explain the preference for a particular junction stacked conformer.     

Solution structure of a viral DNA repair polymerase.

DNA polymerase X (Pol X) from the African swine fever virus (ASFV) specifically binds intermediates in the single-nucleotide base-excision repair process, an activity indicative of repair function. In addition, Pol X catalyzes DNA polymerization with low nucleotide-insertion fidelity. The structural mechanisms by which DNA polymerases confer high or low fidelity in DNA polymerization remain to be elucidated. The three-dimensional structure of Pol X has been determined. Unlike other DNA polymerases, Pol X is formed from only a palm and a C-terminal subdomain. Pol X has a novel palm subdomain fold, containing a positively charged helix at the DNA binding surface. Purine deoxynucleoside triphosphate (dNTP) substrates bind between the palm and C-terminal subdomain, at a dNTP-binding helix, and induce a unique conformation in Pol X. The purine dNTP-bound conformation and high binding affinity for dGTP-Mg(2+) of Pol X may contribute to its low fidelity.     

Crystal structure of an ephrin ectodomain.

Eph receptor tyrosine kinases and their membrane-associated ligands, the ephrins, are essential regulators of axon guidance, cell migration, segmentation, and angiogenesis. There are two classes of vertebrate ephrin ligands which have distinct binding specificities for their cognate receptors. Multimerization of the ligands is required for receptor activation, and ephrin ligands themselves signal intracellularly upon binding Eph receptors. We have determined the structure of the extracellular domain of mouse ephrin-B2. The ephrin ectodomain is an eight-stranded beta barrel with topological similarity to plant nodulins and phytocyanins. Based on the structure, we have identified potential surface determinants of Eph/ephrin binding specificity and a ligand dimerization region. The high sequence similarity among ephrin ectodomains indicates that all ephrins may be modeled upon the ephrin-B2 structure presented here.     

Crystal structure of the processivity clamp loader gamma (gamma) complex of E. coli DNA polymerase III

The gamma complex, an AAA+ ATPase, is the bacterial homolog of eukaryotic replication factor C (RFC) that loads the sliding clamp (beta, homologous to PCNA) onto DNA. The 2.7/3.0 A crystal structure of gamma complex reveals a pentameric arrangement of subunits, with stoichiometry delta':gamma(3):delta. The C-terminal domains of the subunits form a circular collar that supports an asymmetric arrangement of the N-terminal ATP binding domains of the gamma motor and the structurally related domains of the delta' stator and the delta wrench. The structure suggests a mechanism by which the gamma complex switches between a closed state, in which the beta-interacting element of delta is hidden by delta', and an open form similar to the crystal structure, in which delta is free to bind to beta.