The bacterial DNA sliding clamp, ß-clamp: structure, interactions, dynamics and drug discovery.

DNA replication is a tightly coordinated event carried out by a multiprotein replication complex. An essential factor in the bacterial replication complex is the ring-shaped DNA sliding clamp, ß-clamp, ensuring processive DNA replication and DNA repair through tethering of polymerases and DNA repair proteins to DNA. ß -clamp is a hub protein with multiple interaction partners all binding through a conserved clamp binding sequence motif. Due to its central role as a DNA scaffold protein, ß-clamp is an interesting target for antimicrobial drugs, yet little effort has been put into understanding the functional interactions of ß-clamp. In this review, we scrutinize the ß-clamp structure and dynamics, examine how its interactions with a plethora of binding partners are regulated through short linear binding motifs and discuss how contexts play into selection. We describe the dynamic process of clamp loading onto DNA and cover the recent advances in drug development targeting ß-clamp. Despite decades of research in ß-clamps and recent landmark structural insight, much remains undisclosed fostering an increased focus on this very central protein.

Functional hierarchy of PCNA-interacting motifs in DNA processing enzymes.

Numerous eukaryotic DNA processing enzymes, such as DNA polymerases and ligases, bind the processivity factor PCNA, which acts as a platform to recruit and regulate the binding of enzymes to their DNA substrate. Multiple PCNA-interacting motifs (PIPs) are present in these enzymes, but their individual structural and functional role has been a matter of debate. Recent cryo-EM reconstructions of high-fidelity DNA polymerase Pol δ (Pol δ), translesion synthesis DNA polymerase κ (Pol κ) and Ligase 1 (Lig1) bound to a DNA substrate and PCNA demonstrate that the critical interaction with PCNA involves the internal PIP proximal to the catalytic domain. The ancillary PIPs, located in long disordered regions, are instead invisible in the reconstructions, and appear to function as flexible tethers when the enzymes fall off the DNA. In this review, we discuss the recent structural advancements and propose a functional hierarchy for the PIPs in Pol δ, Pol κ, and Lig1.

Coronavirus RNA-dependent RNA polymerase interacts with the p50 regulatory subunit of host DNA polymerase delta and plays a synergistic role with RNA helicase in the induction of DNA damage response and cell cycle arrest in the S phase.

Disruption of the cell cycle is a common strategy shared by many viruses to create a conducible cellular microenvironment for their efficient replication. We have previously shown that infection of cells with gammacoronavirus infectious bronchitis virus (IBV) activated the theataxia-telangiectasia mutated (ATM) Rad3-related (ATR)/checkpoint kinase 1 (Chk1) pathway and induced cell cycle arrest in S and G2/M phases, partially through the interaction of nonstructural protein 13 (nsp13) with the p125 catalytic subunit of DNA polymerase delta (pol δ). In this study, we show, by GST pulldown, co-immunoprecipitation and immunofluorescent staining, that IBV nsp12 directly interacts with the p50 regulatory subunit of pol δ in vitro and in cells overexpressing the two proteins as well as in cells infected with a recombinant IBV harbouring an HA-tagged nsp12. Furthermore, nsp12 from severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 was also able to interact with p50. These interactions play a synergistic role with nsp13 in the induction of S phase arrest. The fact that subunits of an essential cellular DNA replication machinery physically associate with two core replication enzymes from three different coronaviruses highlights the importance of these associations in coronavirus replication and virus-host interaction, and reveals the potential of targeting these subunits for antiviral intervention.

From cue to meaning: The involvement of POLD1 gene in DNA replication, repair and aging.

Aging is an extremely complex biological process. Aging, cancer and inflammation represent a trinity, object of many interesting researches. The accumulation of DNA damage and its consequences progressively interfere with cellular function and increase susceptibility to developing aging condition. DNA Polymerase delta (Pol δ), encoded by POLD1 gene (MIM#174761) on 19q13.3, is well implicated in many steps of the replication program and repair. Thanks to its exonuclease and polymerase activities, the enzyme is involved in the regulation of the cell cycle, DNA synthesis, and DNA damage repair processes. Damaging variants within the exonuclease domain predispose to cancers, while those occurring in the polymerase active site cause the autosomal dominant Progeroid Syndrome called MDPL, Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy Since DNA damage represents the main cause of ageing and age-related pathologies, an overview of critical Pol δ activities will allow to better understand the associations between DNA damage and nearly every aspect of the ageing process, helping the researchers to counteract all the ageing-pathologies at the same time.

Scrutinizing Deleterious Nonsynonymous SNPs and Their Effect on Human POLD1 Gene.

POLD1 (DNA polymerase delta 1, catalytic subunit) is a protein-coding gene that encodes the large catalytic subunit (POLD1/p125) of the DNA polymerase delta (Polδ) complex. The consequence of missense or nonsynonymous SNPs (nsSNPs), which occur in the coding region of a specific gene, is the replacement of single amino acid. It may also change the structure, stability, and/or functions of the protein. Mutation in the POLD1 gene is associated with autosomal dominant predisposition to colonic adenomatous polyps, colon cancer, endometrial cancer (EDMC), breast cancer, and brain tumors. These de novo mutations in the POLD1 gene also result in autosomal dominant MDPL syndrome (mandibular hypoplasia, deafness, progeroid features, and lipodystrophy). In this study, genetic variations of POLD1 which may affect the structure and/or function were analyzed using different types of bioinformatics tools. A total of 17038 nsSNPs for POLD1 were collected from the NCBI database, among which 1317 were missense variants. Out of all missense nsSNPs, 28 were found to be deleterious functionally and structurally. Among these deleterious nsSNPs, 23 showed a conservation scale of >5, 2 were predicted to be associated with binding site formation, and one acted as a posttranslational modification site. All of them were involved in coil, extracellular structures, or helix formation, and some cause the change in size, charge, and hydrophobicity.

The NMR structure of the engineered halophilic DnaE intein for segmental isotopic labeling using conditional protein splicing.

Protein trans-splicing catalyzed by split inteins has been used for segmental isotopic labeling of proteins for alleviating the complexity of NMR signals. Whereas inteins spontaneously trigger protein splicing upon protein folding, inteins from extremely halophilic organisms require a high salinity condition to induce protein splicing. We designed and created a salt-inducible intein from the widely used DnaE intein from Nostoc punctiforme by introducing 29 mutations, which required a lower salt concentration than naturally occurring halo-obligate inteins. We determined the NMR solution structure of the engineered salt-inducible DnaE intein in 2 M NaCl, showing the essentially identical three-dimensional structure to the original one, albeit it unfolds without salts. The NMR structure of a halo-obligate intein under high salinity suggests that the stabilization of the active folded conformation is not a mere result of various intramolecular interactions but the subtle energy balance from the complex interactions, including the solvation energy, which involve waters, ions, co-solutes, and protein polypeptide chains.

Solving the enigma of POLD1 p.V295M as a potential cause of increased cancer risk.

Germline variants that affect the proofreading activity of polymerases epsilon (POLE) and delta (POLD1) predispose to colorectal adenomas and carcinomas, among other cancers. All cancer-associated pathogenic variants reported to date consist of non-disruptive genetic changes affecting the sequence that codifies the exonuclease domain (ED). Generally, disruptive (frameshift, stop-gain) POLE and POLD1 variants and missense variants outside the ED do not predispose to cancer. However, this statement may not be true for some, very specific variants that would indirectly affect the proofreading activity of the corresponding polymerase. We evaluated, by using multiple approaches, the possibility that POLD1 c.883G>A; p.(Val295Met), -a variant located 9 amino acids upstream the ED and present in ~0.25% of hereditary cancer patients-, affects POLD1 proofreading activity. Our findings show cumulative evidence that support no alteration of the proofreading activity and lack of association with cancer. The variant is classified as likely benign according to the ACMG/AMP guidelines.

The stabilized Pol31-Pol3 interface counteracts Pol32 ablation with differential effects on repair.

DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of pol31 is lethal, and compromising the Pol3-Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying pol32Δ. We have identified alanine-substitutions in pol31 that suppress these deficiencies in pol32Δ cells. We characterize two mutants, pol31-T415A and pol31-W417A, which map to a solvent-exposed loop that mediates Pol31-Pol3 and Pol31-Rev3 interactions. The pol31-T415A substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as pol31-T415A, leads to reduced Pol3 and Pol31 protein levels, which are restored by pol31-W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in pol32Δ cells is not restored by pol31-W417A, highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that pol32Δ and pol31-T415A indirectly destabilize DNA pol α and pol ε at stalled forks.

Coupling of the engineered DNA "mutator" to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces.

DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these 'mutators' was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10-5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.

Novel Escherichia coli active site dnaE alleles with altered base and sugar selectivity.

The Escherichia coli dnaE gene encodes the α-catalytic subunit (pol IIIα) of DNA polymerase III, the cell's main replicase. Like all high-fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so-called "steric gate" residue in the active site of the polymerase that physically clashes with the 2'-OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α-θ-ε subunits). Of the possible 38 mutants, only nine were successfully sub-cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid-encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature.

Analytical Ultracentrifugation for Analysis of Protein-Nucleic Acid Interactions.

Analytical ultracentrifugation is a powerful tool to characterize interactions of macromolecules in solution. In sedimentation velocity experiments, the sedimentation of interaction partners and complexes can be monitored directly and can be used to characterize interactions quantitatively. As an example, we show how the interaction of the clamp loader subcomplex of DNA polymerase III from E. coli and a template/primer DNA saturated with single-stranded DNA-binding protein can be analyzed by analytical ultracentrifugation with fluorescence detection.

DNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen.

The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD-primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.

Structural Mechanisms for Replicating DNA in Eukaryotes.

The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein-nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.

Biochemical characterization of a unique DNA polymerase A from the extreme radioresistant organism Deinococcus radiodurans.

Deinococcus radiodurans survives extraordinary doses of ionizing radiation and desiccation that cause numerous DNA strand breaks. D. radiodurans DNA polymerase A (DrPolA) is essential for reassembling the shattered genome, while its biochemical property has not been fully demonstrated. In this study, we systematically examined the enzymatic activities of DrPolA and characterized its unique features. DrPolA contains an N-terminal nuclease domain (DrPolA-NTD) and a C-terminal Klenow fragment (KlenDr). Compared with the Klenow fragment of E. coli Pol I, KlenDr shows higher fidelity despite the lacking of 3'-5' exonuclease proofreading activity and prefers double-strand DNA rather than Primer-Template substrates. Apart from the well-annotated 5'-3' exonuclease and flap endonuclease activities, DrPolA-NTD displays approximately 140-fold higher gap endonuclease activity than its homolog in E. coli and Human FEN1. Its 5'-3' exonuclease activity on ssDNA, gap endonuclease, and Holliday junction cleavage activities are greatly enhanced by Mn2+. The DrPolA-NTD deficient strain shows increased sensitivity to UV and gamma-ray radiation. Collectively, our results reveal distinct biochemical characteristics of DrPolA during DNA degradation and re-synthesis, which provide new insight into the outstanding DNA repair capacity of D. radiodurans.

Transcription activation by a sliding clamp.

Transcription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator gp33, and a universal component of cellular DNA replication, the sliding clamp gp45. Although genetic and biochemical studies have elucidated many aspects of T4 late gene transcription, no precise structure of the transcription machinery in the process is available. Here, we report the cryo-EM structures of a gp55-dependent RNAP-promoter open complex and an intact gp45-dependent transcription activation complex. The structures reveal the interactions between gp55 and the promoter DNA that mediate the recognition of T4 late promoters. In addition to the σR2 homology domain, gp55 has a helix-loop-helix motif that chaperons the template-strand single-stranded DNA of the transcription bubble. Gp33 contacts both RNAP and the upstream double-stranded DNA. Gp45 encircles the DNA and tethers RNAP to it, supporting the idea that gp45 switches the promoter search from three-dimensional diffusion mode to one-dimensional scanning mode.

Underappreciated Roles of DNA Polymerase δ in Replication Stress Survival.

Recent structural analysis of Fe-S centers in replication proteins and insights into the structure and function of DNA polymerase δ (DNA Pol δ) subunits have shed light on the key role played by this polymerase at replication forks under stress. The sequencing of cancer genomes reveals multiple point mutations that compromise the activity of POLD1, the DNA Pol δ catalytic subunit, whereas the loci encoding the accessory subunits POLD2 and POLD3 are amplified in a very high proportion of human tumors. Consistently, DNA Pol δ is key for the survival of replication stress and is involved in multiple long-patch repair pathways. Synthetic lethality arises from compromising the function and availability of the noncatalytic subunits of DNA Pol δ under conditions of replication stress, opening the door to novel therapies.

Structural, Dynamic, and Functional Characterization of a DnaX Mini-intein Derived from Spirulina platensis Provides Important Insights into Intein-Mediated Catalysis of Protein Splicing.

Protein splicing is a self-catalyzed post-translational modification in which the intein enzyme excises itself from a precursor protein and ligates the flanking sequences to produce a mature protein. We report the solution structure of a 136-residue DnaX mini-intein enzyme derived from the cyanobacterium Spirulina platensis. This sequence adopts a well-defined globular structure and forms a horseshoe-shaped fold commonly found in the HINT (hedgehog intein) topology. Backbone dynamics and hydrogen exchange experiments revealed conserved motions on various time scales, which is proposed to be a characteristic of the intein fold. Interestingly, several dynamic motions were found in symmetrically equivalent positions within the protein structure, which might be a consequence of the symmetrical intein fold. In cell splicing activity showed that Spl DnaX mini-intein is a highly active enzyme. The precursor protein was not detected at any timepoint of the assay. Apart from the splicing reaction, catalytic cleavage at the N- and C-termini of the precursor protein was also observed. To determine the roles of the catalytic residues in splicing and cleavage reactions, all combinations of alanine mutations of these residues were generated and functionally characterized. This in-depth analysis revealed cooperativity between these catalytic residues, which suppresses the N- and C-terminal cleavage reactions and enhances the yield of the spliced product. Overall, this study provides a thorough structural, dynamic, and functional characterization of a new intein sequence and adds to the collection of these unique enzymes that have found tremendous applications in biochemistry and biotechnology.

Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA.

The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Šcryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.

Structure and mechanism of B-family DNA polymerase ζ specialized for translesion DNA synthesis.

DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31' Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein-protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ's role in TLS and provide a framework for new cancer therapeutics.