Human Primase Research Articles

The role of catalytic and regulatory domains of human PrimPol in DNA binding and synthesis.

Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.

The DNA ligands Arg47 and Arg76 are crucial for catalysis by human PrimPol

Human primase and DNA polymerase PrimPol re-starts stalled replication forks by repriming downstream DNA lesions and protects cells against DNA damage. Structure of the catalytic core of PrimPol with DNA primer, template and incoming dATP was solved but the mechanisms of DNA polymerase and primase activities of PrimPol are not fully understood. In this work, using site-directed mutagenesis we biochemically analyzed the role of active site residues Arg47 and Arg76 contacting DNA template in DNA polymerase and primase activities of PrimPol. The substitution R47A diminished the DNA polymerase and primase activities of PrimPol whereas the single amino acid substitution R76A caused almost complete loss of catalytic activities. Both amino acid substitutions affected the spectrum of dNMPs incorporation on undamaged DNA templates and opposite 8-oxoguanine. Finally, substitutions of the Arg47 and Arg76 residues attenuated the formation of the stable PrimPol:DNA complex in the presence of ATP/dNTPs. Together, these findings suggest a key role of the Arg47 and Arg76 in DNA synthesis by PrimPol.

Evaluation of potential drugs against leishmaniasis targeting catalytic subunit of Leishmania donovani nuclear DNA primase using ligand based virtual screening, docking and molecular dynamics approaches

Leishmania donovani, causes leishmaniasis, a global health trouble with around 89 different countries and its population under its risk. Replication initiation events have been instrumental in regulating the DNA duplication and as the small subunit of L. donovani nuclear DNA primase (Ld-PriS) inherits the catalytic site, it plays a vital role in DNA replication. In this study we have aimed Ld-PriS for the first time as a prospective target for the application of drug against Leishmania parasite. 3-D structures of Ld-PriS were built and ligand-based virtual screening was performed using hybrid similarity recognition techniques. Ligands from the ZINC database were used for the screening purposes based on known DNA primase inhibitor Sphingosine as a query. Top 150 ligands were taken into consideration for molecular docking against the query protein (Ld-PriS) using PyRx and iGEMDOCK softwares. Top five compounds with the best docking score were selected for pharmacokinetic investigation and molecular dynamic simulation. These top five screened inhibitors showed very poor binding affinity toward the catalytic subunit of human primase indicating their safety toward the host normal replication mechanism. The top five compounds showed good pharmacokinetic profiles and ADMET predictions revealed good absorption, solubility, permeability, uniform distribution, proper metabolism, minimal toxicity and good bioavailability. Simulation studies upto 50 ns revealed the three leads ZINC000009219046, ZINC000025998119 and ZINC000004677901 bind with Ld-PriS throughout the simulation and there were no huge variations in their backbone suggesting that these three may play as potential lead compounds for developing new drug against leishmaniasis. Communicated by Ramaswamy H. Sarma

Mutation effects on charge transport through the p58c iron-sulfur protein.

Growing experimental evidence indicates that iron–sulfur proteins play key roles in DNA repair and replication. In particular, charge transport between [Fe4S4] clusters, mediated by proteins and DNA, may convey signals to coordinate enzyme action. Human primase is a well studied [Fe4S4] protein, and its p58c domain (which contains an [Fe4S4] cluster) plays a role in the initiation of DNA replication. The Y345C mutation in p58c is linked to gastric tumors and may influence the protein-mediated charge transport. The complexity of protein–DNA systems, and the intricate electronic structure of [Fe4S4] clusters, have impeded progress into understanding functional charge transport in these systems. In this study, we built force fields to describe the high potential [Fe4S4] cluster in both oxidation states. The parameterization is compatible with AMBER force fields and enabled well-balanced molecular dynamics simulations of the p58c–RNA/DNA complex relevant to the initiation of DNA replication. Using the molecular mechanics Poisson–Boltzmann and surface area solvation method on the molecular dynamics trajectories, we find that the p58c mutation induces a modest change in the p58c–duplex binding free energy in agreement with recent experiments. Through kinetic modeling and analysis, we identify key features of the main charge transport pathways in p58c. In particular, we find that the Y345C mutation partially changes the composition and frequency of the most efficient (and potentially relevant to the biological function) charge transport pathways between the [Fe4S4] cluster and the duplex. Moreover, our approach sets the stage for a deeper understanding of functional charge transfer in [Fe4S4] protein–DNA complexes.

Structural Basis for Inhibition of Human Primase by Arabinofuranosyl Nucleoside Analogues Fludarabine and Vidarabine.

Nucleoside analogues are widely used in clinical practice as chemotherapy drugs. Arabinose nucleoside derivatives such as fludarabine are effective in the treatment of patients with acute and chronic leukemias and non-Hodgkin’s lymphomas. Although nucleoside analogues are generally known to function by inhibiting DNA synthesis in rapidly proliferating cells, the identity of their in vivo targets and mechanism of action are often not known in molecular detail. Here we provide a structural basis for arabinose nucleotide-mediated inhibition of human primase, the DNA-dependent RNA polymerase responsible for initiation of DNA synthesis in DNA replication. Our data suggest ways in which the chemical structure of fludarabine could be modified to improve its specificity and affinity toward primase, possibly leading to less toxic and more effective therapeutic agents.

Yeast require redox switching in DNA primase

Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S]+ species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.

Structural and biochemical insights into inhibition of human primase by citrate

The eukaryotic primase/polymerase complex synthesizes approximately 107 primers, one per Okazaki fragment, during the replication of mammalian chromosomes, which contain 109 base pairs. Primase catalyzes the synthesis of a short RNA segment to a single-stranded DNA template. Primase is important in DNA replication because no known replicative DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA primer. The primase subcomplex is composed of a small catalytic subunit (p49), and a large accessory subunit (p58). Priming mechanisms remain poorly understood, although large numbers of structures of archaeal and eukaryotic p49 and/or p58 as well as structures of bacterial enzymes have been determined. In this study, we determined the structure of human p49 at 2.2 Å resolution with citrate in its inactive forms. Dibasic citrate was bound at the nucleotide triphosphate (NTP) β, γ-phosphate binding site through nine hydrogen bonds. We also measured the dissociation constant of citrate and NTPs. We further demonstrated that the p49 activity is regulated by pH and citrate, which was not previously recognized as a key regulator of DNA replication. We propose that the citrate inhibits the primase and regulates DNA replication at the replication fork.

DnaG Primase-A Target for the Development of Novel Antibacterial Agents.

The bacterial primase—an essential component in the replisome—is a promising but underexploited target for novel antibiotic drugs. Bacterial primases have a markedly different structure than the human primase. Inhibition of primase activity is expected to selectively halt bacterial DNA replication. Evidence is growing that halting DNA replication has a bacteriocidal effect. Therefore, inhibitors of DNA primase could provide antibiotic agents. Compounds that inhibit bacterial DnaG primase have been developed using different approaches. In this paper, we provide an overview of the current literature on DNA primases as novel drug targets and the methods used to find their inhibitors. Although few inhibitors have been identified, there are still challenges to develop inhibitors that can efficiently halt DNA replication and may be applied in a clinical setting.

In vitro lesion bypass by human PrimPol

Many human DNA polymerases bypass DNA damage during translesion synthesis (TLS). Human primase and polymerase, PrimPol, assists fork progression by repriming DNA synthesis downstream of the lesion using its DNA primase activity. We tested the properties of PrimPol as a TLS polymerase in the presence of different metal ions in vitro. We demonstrate that in the presence of Mg2+ ions PrimPol carries out efficient and relatively accurate synthesis past 8-oxoguanine and 5-formyluracil. It also bypasses an abasic site and O6-methylguanine, but is blocked by thymine glycol and 1,N6-ethenoadenine. Substitution of Mg2+ with Mn2+ stimulates the TLS activity of PrimPol and allows for efficient, but error-prone, synthesis on DNA templates containing all tested DNA lesions, including thymine glycol and 1,N6-ethenoadenine. The TLS activity of PrimPol has possible relevant functions in vivo; e.g., the combined primase and DNA polymerase activities of PrimPol might facilitate replication of DNA with clustered damage.

Cracking Open a Molecular Calculator: DNA Charge Transport and Primase

De novo initiation of DNA synthesis by DNA polymerases requires formation of an initial “primer” on the template. In eukaryotes, this function is provided by a distinct DNA primase. Primase, a DNA-dependent RNA polymerase, generates short (8-12 nt) RNA primers on ssDNA templates. These initial RNA primers are handed off to DNA polymerase α (pol α), which extends the primer by ∼20 nts. This primed RNA/DNA substrate is then handed off to processive polymerases e or δ, which synthesize the vast majority of new DNA on the leading and lagging strands. X-ray crystal structures of human primase have been used to propose models for the catalytic mechanism. However, the molecular basis for primer counting and handoff remains unclear. Primase contains a 4Fe-4S cluster in the p58C domain critical to pol-prim loading and activity, but its specific function is not known. We are investigating the hypothesis that the function of the cluster is to drive primer length counting. Well-stacked duplex DNA has been shown capable of transporting charge over long distances, and it has been proposed this phenomenon enables communication between Fe-S cluster-containing proteins. In this study, we show the 4Fe-4S cluster in wild-type p58C is capable of mediating charge transport (CT) with DNA. DNA-modified electrodes were used to characterize the electrochemistry of p58C. Based on previously determined WT structures, we identified a possible pathway from the cluster to the DNA binding site in p58C and designed mutants of key residues in this pathway. Mutation of Tyr345 to Phe greatly diminishes DNA CT. Primase activity assays with WT and mutant proteins revealed substantial differences in product length distribution. These data suggest charge transport is involved in primer length counting and may signal for primer template handoff to pol α.

Crystal Structure of the Human Primase

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme's conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.

Herpesviral helicase-primase subunit UL8 is inactivated B-family polymerase.

Herpesviruses are large DNA viruses causing a variety of diseases in humans and animals. To develop effective treatment, it is important to understand the mechanisms of their replication. One of the components of the herpesviral DNA replication system is a helicase-primase complex, consisting of UL5 (helicase), UL52 (primase) and UL8. UL8 is an essential herpesviral protein involved in multiple protein-protein interactions. Intriguingly, so far no UL8 homologs outside of herpesviruses could be identified. Moreover, nothing is known about its structure or domain organization. Here, combining sensitive homology detection methods and homology modeling, we found that the UL8 protein family is related to B-family polymerases. In the course of evolution, UL8 has lost the active site and has undergone a reduction of DNA-binding motifs. The loss of active site residues explains the failure to detect any catalytic activity of UL8. A structural model of human herpes virus 1 UL8 constructed as part of the study is consistent with the mutation data targeting its interaction with primase UL52. It also provides a platform for studying multiple interactions that UL8 is involved in. The two other components of helicase-primase complex show evolutionary links with a newly characterized human primase that also has DNA polymerase activity (PrimPol) and the Pif1 helicase, respectively. The role of these enzymes in recovering stalled replication forks suggests mechanistic and functional similarities with herpesviral proteins. venclovas@ibt.lt Supplementary data are available at Bioinformatics online.

Crystallization and preliminary X-ray diffraction analysis of human DNA primase

Human primase synthesizes RNA primers and transfers them to the active site of Pol α with subsequent extension with dNTPs. Human primase is a heterodimer of two subunits: a small catalytic subunit (p49) and a large subunit (p58). The structural details of the initiation and elongation steps of primer synthesis, as well as primer length counting, are not known. To address these questions, structural studies of human primase were initiated. Two types of crystals were obtained. The best diffracting crystals belonged to space group P1, with unit-cell parameters a = 86.2, b = 88.9, c = 94.68 Å, α = 93.82, β = 96.57, γ = 111.72°, and contained two heterodimers of full-length p49 and p59 subunits in the asymmetric unit.

PrimPol breaks replication barriers

Faithful bypass of replication forks encountering obstructive DNA lesions is essential to prevent fork collapse and cell death. PrimPol is a new human primase and translesion polymerase that is able to bypass fork-blocking UV-induced lesions and to restart replication by origin-independent repriming.

Repriming of DNA synthesis at stalled replication forks by human PrimPol

DNA replication forks that collapse during the process of genomic duplication lead to double-strand breaks and constitute a threat to genomic stability. The risk of fork collapse is higher in the presence of replication inhibitors or after UV irradiation, which introduces specific modifications in the structure of DNA. In these cases, fork progression may be facilitated by error-prone translesion synthesis (TLS) DNA polymerases. Alternatively, the replisome may skip the damaged DNA, leaving an unreplicated gap to be repaired after replication. This mechanism strictly requires a priming event downstream of the lesion. Here we show that PrimPol, a new human primase and TLS polymerase, uses its primase activity to mediate uninterrupted fork progression after UV irradiation and to reinitiate DNA synthesis after dNTP depletion. As an enzyme involved in tolerance to DNA damage, PrimPol might become a target for cancer therapy.

Insights into Eukaryotic Primer Synthesis from Structures of the p48 Subunit of Human DNA Primase

DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8–10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn2+ but not Mg2+ significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn2+ provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.

Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering

Initiation of DNA synthesis in genomic duplication depends on primase, the DNA-dependent RNA polymerase that synthesizes de novo the oligonucleotides that prime DNA replication. Due to the discontinuous nature of DNA replication, primase activity on the lagging strand is required throughout the replication process. In eukaryotic cells, the presence of primase at the replication fork is secured by its physical association with DNA polymerase α (Pol α), which extends the RNA primer with deoxynucleotides. Our knowledge of the mechanism that primes DNA synthesis is very limited, as structural information for the eukaryotic enzyme has proved difficult to obtain. Here, we describe the crystal structure of human primase in heterodimeric form consisting of full-length catalytic subunit and a C-terminally truncated large subunit. We exploit the crystallographic model to define the architecture of its nucleotide elongation site and to show that the small subunit integrates primer initiation and elongation within the same set of functional residues. Furthermore, we define in atomic detail the mode of association of primase to Pol α, the critical interaction that keeps primase tethered to the eukaryotic replisome.

Crystal structure of the C-terminal domain of human DNA primase large subunit

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes’ large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.

Mechanisms by which human DNA primase chooses to polymerize a nucleoside triphosphate.

Human DNA primase synthesizes short RNA primers that DNA polymerase alpha then elongates during the initiation of all new DNA strands. Even though primase misincorporates NTPs at a relatively high frequency, this likely does not impact the final DNA product since the RNA primer is replaced with DNA. We used an extensive series of purine and pyrimidine analogues to provide further insights into the mechanism by which primase chooses whether or not to polymerize a NTP. Primase readily polymerized a size-expanded cytosine analogue, 1,3-diaza-2-oxophenothiazine NTP, across from a templating G but not across from A. The enzyme did not efficiently polymerize NTPs incapable of forming two Watson-Crick hydrogen bonds with the templating base with the exception of UTP opposite purine deoxyribonucleoside. Likewise, primase did not generate base pairs between two nucleotides with altered Watson-Crick hydrogen-bonding patterns. Examining the mechanism of NTP polymerization revealed that human primase can misincorporate NTPs via both template misreading and a primer-template slippage mechanism. Together, these data demonstrate that human primase strongly depends on Watson-Crick hydrogen bonds for efficient nucleotide polymerization, much more so than the mechanistically related herpes primase, and provide insights into the potential roles of primer-template stability and base tautomerization during misincorporation.

Discovery of a novel series of inhibitors of human cytomegalovirus primase

Infection by human cytomegalovirus (hCMV) remains a potent threat to susceptible people throughout the world. We have discovered a series of imidazolyl-pyrimidine compounds, which were found to be irreversible inhibitors of the hCMV UL70 primase based on results from radiolabeling and SAR studies. Two promising analogs are described that rival ganciclovir and cidofovir in antiviral potency and possess improved cytotoxicity profiles.