The conserved RNP motif of the herpes simplex virus 1 family B DNA polymerase is crucial for viral DNA synthesis but not polymerase activity.

The herpes simplex virus 1 DNA polymerase contains a highly conserved structural motif found in most family B polymerases and certain RNA-binding proteins. To investigate its importance within cells, we constructed a mutant virus with substitutions in two residues of the motif and a rescued derivative. The substitutions resulted in severe impairment of plaque formation, yields of infectious virus, and viral DNA synthesis while not meaningfully affecting expression of the mutant enzyme, its co-localization with the viral single-stranded DNA binding protein at intranuclear punctate sites in non-complementing cells or in replication compartments in complementing cells, or viral DNA polymerase activity. Taken together, our results indicate that the RNA binding motif plays a crucial role in herpes simplex virus 1 DNA synthesis through a mechanism separate from effects on polymerase activity, thus identifying a distinct essential function of this motif with implications for hypotheses regarding its biochemical functions.

CryoEM insights into RNA primer synthesis by the human primosome.

Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo-electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation.

CST-polymerase α-primase solves a second telomere end-replication problem.

Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres1, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand DNA synthesis ('the end-replication problem'2). Here we report a second end-replication problem that originates from the incomplete duplication of the C-rich telomeric repeat strand (C-strand) by lagging-strand DNA synthesis. This problem is resolved by fill-in synthesis mediated by polymerase α-primase bound to Ctc1-Stn1-Ten1 (CST-Polα-primase). In vitro, priming for lagging-strand DNA replication does not occur on the 3' overhang and lagging-strand synthesis stops in a zone of approximately 150 nucleotides (nt) more than 26 nt from the end of the template. Consistent with the in vitro data, lagging-end telomeres of cells lacking CST-Polα-primase lost 50-60 nt of telomeric CCCTAA repeats per population doubling. The C-strands of leading-end telomeres shortened by around 100 nt per population doubling, reflecting the generation of 3' overhangs through resection. The measured overall C-strand shortening in the absence of CST-Polα-primase fill-in is consistent with the combined effects of incomplete lagging-strand synthesis and 5' resection at the leading ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-rich strand and CST-Polα-primase to maintain the C-strand.

DNA Polymerase I Large Fragment from Deinococcus radiodurans, a Candidate for a Cutting-Edge Room-Temperature LAMP.

In recent years, the loop-mediated isothermal amplification (LAMP) technique, designed for microbial pathogen detection, has acquired fundamental importance in the biomedical field, providing rapid and precise responses. However, it still has some drawbacks, mainly due to the need for a thermostatic block, necessary to reach 63 °C, which is the BstI DNA polymerase working temperature. Here, we report the identification and characterization of the DNA polymerase I Large Fragment from Deinococcus radiodurans (DraLF-PolI) that functions at room temperature and is resistant to various environmental stress conditions. We demonstrated that DraLF-PolI displays efficient catalytic activity over a wide range of temperatures and pH, maintains its activity even after storage under various stress conditions, including desiccation, and retains its strand-displacement activity required for isothermal amplification technology. All of these characteristics make DraLF-PolI an excellent candidate for a cutting-edge room-temperature LAMP that promises to be very useful for the rapid and simple detection of pathogens at the point of care.

Innovative Highly Specific Nucleic Acid Isothermal Detection Assay Based on the Polymerization-Coupled Endonuclease Activity of Prokaryotic DNA Polymerase I.

In this study, we developed an innovative highly specific nucleic acid isothermal detection assay based on prokaryotic DNA polymerase I with exquisitely designed fluorescent probes, achieving high sensitivity and 100% specificity within 30 min. The fluorescent nucleic acid probe was designed and constructed based on the specific flap cleavage endonuclease activity of prokaryotic DNA polymerase I (including the Bst, Bsu, Bsm, and Klenow DNA polymerases). The flap endonuclease activity depends on the length of the flap DNA and polymerization activity, which greatly reduces the false-positive rate caused by primer dimerization. This robust assay was also validated by the detection of rotavirus with great specificity and sensitivity. It could be a great alternative to qPCR in the field of point-of-care detection of pathogens.

One-Pot Enzymatic Preparation of Oligonucleotides with an Expanded Genetic Alphabet via Controlled Pause and Restart of Primer Extension: Making Unnatural Out of Natural.

The genetic alphabet of life has been dramatically expanded via the development of unnatural base pairs (UBPs) that work as efficiently as natural base pairs in the storage and retrieval of genetic information. Among the most predominant UBPs, dNaM-dTPT3 and its analogues have been successfully employed to build semisynthetic cells with a functional six-letter genome. With the rapidly growing applications of UBPs in vitro and in vivo, there is an ever-increasing demand for DNA oligonucleotides containing unnatural bases (UBs) at desired positions. Conventional solid-phase synthesis of oligonucleotides has intrinsic limitations and needs to use unstable unnatural phosphoramidites and a DNA synthesizer, so it does not meet the daily urgent requirement for a few UB-containing DNA oligonucleotides in the laboratory. In this work, we develop a one-pot enzymatic method for preparing dNaM- or dTPT3-containing DNA oligonucleotides via controlled pause and restart of primer extension mediated by Klenow fragment (exo-). By systematic optimization of the reaction conditions, high efficiencies and product purities have been achieved. The universality of this method for preparing DNA oligonucleotides containing dNaM or dTPT3 in different sequence contexts is also demonstrated. This method allows convenient production of an arbitrary UB-containing DNA oligonucleotide in a single test tube with only two natural DNA oligonucleotides, stable nucleoside triphosphates, Klenow fragment (exo-), and other common reagents in the laboratory, providing the lowest cost and the highest simplicity for the enzymatic preparation of UB-containing oligonucleotides. Clearly, this method has great potential to facilitate the in vitro and in vivo applications of the UBPs.

The Antitumor Effect of the DNA Polymerase Alpha Inhibitor ST1926 in Glioblastoma: A Proteomics Approach.

Glioblastoma Multiforme (GBM) is the most aggressive form of malignant brain tumor. The median survival rate does not exceed two years, indicating an imminent need to develop novel therapies. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types. We have shown that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1), which is involved in initiating DNA synthesis in eukaryotic cells. POLA1 levels are elevated in GBM versus normal brain tissues. Therefore, we studied the antitumor effects of ST1926 in several human GBM cell lines. We further explored the global protein expression profiles in GBM cell lines using liquid chromatography coupled with tandem mass spectrometry to identify new targets of ST1926. Low sub-micromolar concentrations of ST1926 potently decreased cell viability, induced cell damage and apoptosis, and reduced POLA1 protein levels in GBM cells. The proteomics profiles revealed 197 proteins significantly differentially altered upon ST1926 treatment of GBM cells involved in various cellular processes. We explored the differential gene and protein expression of significantly altered proteins in GBM compared to normal brain tissues.

The Saccharomyces cerevisiae mitochondrial DNA polymerase and its contribution to the knowledge about human POLG-related disorders.

Most eukaryotes possess a mitochondrial genome, called mtDNA. In animals and fungi, the replication of mtDNA is entrusted by the DNA polymerase γ, or Pol γ. The yeast Pol γ is composed only of a catalytic subunit encoded by MIP1. In humans, Pol γ is a heterotrimer composed of a catalytic subunit homolog to Mip1, encoded by POLG, and two accessory subunits. In the last 25 years, more than 300 pathological mutations in POLG have been identified as the cause of several mitochondrial diseases, called POLG-related disorders, which are characterized by multiple mtDNA deletions and/or depletion in affected tissues. In this review, at first, we summarize the biochemical properties of yeast Mip1, and how mutations, especially those introduced recently in the N-terminal and C-terminal regions of the enzyme, affect the in vitro activity of the enzyme and the in vivo phenotype connected to the mtDNA stability and to the mtDNA extended and point mutability. Then, we focus on the use of yeast harboring Mip1 mutations equivalent to the human ones to confirm their pathogenicity, identify the phenotypic defects caused by these mutations, and find both mechanisms and molecular compounds able to rescue the detrimental phenotype. A closing chapter will be dedicated to other polymerases found in yeast mitochondria, namely Pol ζ, Rev1 and Pol η, and to their genetic interactions with Mip1 necessary to maintain mtDNA stability and to avoid the accumulation of spontaneous or induced point mutations.

Investigation of thermal stability characteristic in family A DNA polymerase - A theoretical study.

DNA polymerases create complementary DNA strands in living cells and are crucial to genome transmission and maintenance. These enzymes possess similar human right-handed folds which contain thumb, fingers, and palm subdomains and contribute to polymerization activities. These enzymes are classified into seven evolutionary families, A, B, C, D, X, Y, and RT, based on amino acid sequence analysis and biochemical characteristics. Family A DNA polymerases exist in an extended range of organisms including mesophilic, thermophilic, and hyper-thermophilic bacteria, participate in DNA replication and repair, and have a broad application in molecular biology and biotechnology. In this study, we attempted to detect factors that play a role in the thermostability properties of this family member despite their remarkable similarities in structure and function. For this purpose, similarities and differences in amino acid sequences, structure, and dynamics of these enzymes have been inspected. Our results demonstrated that thermophilic and hyper-thermophilic enzymes have more charged, aromatic, and polar residues than mesophilic ones and consequently show further electrostatic and cation-pi interactions. In addition, in thermophilic enzymes, aliphatic residues tend to position in buried states more than mesophilic enzymes. These residues within their aliphatic parts increase hydrophobic core packing and therefore enhance the thermostability of these enzymes. Furthermore, a decrease in thermophilic cavities volumes assists in the protein compactness enhancement. Moreover, molecular dynamic simulation results revealed that increasing temperature impacts mesophilic enzymes further than thermophilic ones that reflect on polar and aliphatic residues surface area and hydrogen bonds changes.

Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach.

The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme. In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP. The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme. The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated. Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.

Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system.

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.

Human DNA polymerase α has a strong mutagenic potential at the initial steps of DNA synthesis.

DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19 Šresolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Šresolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

Recognition of a Clickable Abasic Site Analog by DNA Polymerases and DNA Repair Enzymes.

Azide-alkyne cycloaddition ("click chemistry") has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the "A-rule", directing residual but easily detectable incorporation of dAMP by E. coli DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase ß. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and E. coli AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.

A Xp22.11-p21.3 microdeletion in a three-generation family supports male lethality of POLA1 nullisomy resulting in reduced fertility of female carriers.

POLA1 encodes a subunit of the DNA polymerase alpha, a key enzyme for the initiation of DNA synthesis. In males, hemizygous hypomorphic variants in POLA1 have been identified as the cause of X-linked pigmentary reticulate disorder (XLPDR) and a novel X-linked neurodevelopmental disorder termed Van Esch-O'Driscoll syndrome (VEODS), while female carriers have been reported to be healthy. Nullisomy for POLA1 was speculated to be lethal due to its crucial function, while the effect of loss of one allele in females remained unknown. Here, we report on a three-generation family harboring a deletion of POLA1 in females showing subfertility as the only phenotype. Our findings show that heterozygous deletions or truncating variants in females with skewed X inactivation do not cause VEODS and support the hypothesis of very early embryonic lethality in males with POLA1 nullisomy.

DarT-mediated mtDNA damage induces dynamic reorganization and selective segregation of mitochondria.

Mitochondria are dynamic organelles that play essential roles in cell growth and survival. Processes of fission and fusion are critical for the distribution, segregation, and maintenance of mitochondria and their genomes (mtDNA). While recent work has revealed the significance of mitochondrial organization for mtDNA maintenance, the impact of mtDNA perturbations on mitochondrial dynamics remains less understood. Here, we develop a tool to induce mitochondria-specific DNA damage using a mitochondrial-targeted base modifying bacterial toxin, DarT. Following damage, we observe dynamic reorganization of mitochondrial networks, likely driven by mitochondrial dysfunction. Changes in the organization are associated with the loss of mtDNA, independent of mitophagy. Unexpectedly, perturbation to exonuclease function of mtDNA replicative polymerase, Mip1, results in rapid loss of mtDNA. Our data suggest that, under damage, partitioning of defective mtDNA and organelle are de-coupled, with emphasis on mitochondrial segregation independent of its DNA. Together, our work underscores the importance of genome maintenance on mitochondrial function, which can act as a modulator of organelle organization and segregation.

Potential G-quadruplexes within the Promoter Nuclease Hypersensitive Sites of the Heat-Responsive Genes in Rice.

G-quadruplexes (G4s) have been shown to be involved in the regulation of multiple cellular processes. Exploring putative G4-forming sequences (PQSs) in heat-responsive genes of rice and their folding structures under different conditions will help to understand the mechanism in response to heat stress. In this work, we discovered a prevalence of PQSs in nuclease hypersensitive sites within the promoters of heat-responsive genes. Moreover, 50 % of the searched G3 PQSs ((G3+ L1-7 )3+ G3+ ) locate in heat shock transcription factors. Circular dichroism spectroscopy, thermal difference spectroscopy, and UV melting analysis demonstrated the representative PQSs could adopt stable G4s at physiological temperature and potassium concentration. These PQSs were able to stall Klenow fragment (KF) DNA polymerase by the formation of G4s. However, the G4s with Tm values around 50-60 °C could be increasingly unwound by KF with the increase of temperatures from 25 to 50 °C, implying that these G4s could sense the changes in temperature by structural switch. This work offers fresh clues to understanding the potential of G4-involved functions of PQSs and the molecular events in plants in response to heat stress.

Solid-state nanopore analysis on the conformation change of DNA polymerase I induced by a DNA substrate.

Proteins with a changeable conformation, such as polymerases, play a very important role in various life activities. Their conformational changes can be reflected in their structural size and flexibility, which may influence their transport kinetics. Recently, solid-state nanopore sensors have been widely applied to characterize the conformation of proteins and other complex structures as sensitive and high throughput single-molecule detectors. In this work, we used a SiN nanopore sensor to study the conformational changes between the Klenow fragment (KF) and its monomer complex with a DNA substrate (KF-DNA). By calculating their hydrodynamic radii, pore volume, the duration of translocation events, drift velocity, and molecular dynamics simulations, we found that the KF-DNA monomer complex has a tighter structure and transports slower. The study performed here can be potentially used to identify single polymerases in real time and may ultimately reveal conformation changes and the interaction between polymerases and their substrates.

Insight into RNA-DNA primer length counting by human primosome.

The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), synthesizes chimeric RNA-DNA primers of a limited length for DNA polymerases delta and epsilon to initiate DNA replication on both chromosome strands. Despite recent structural insights into the action of its two catalytic centers, the mechanism of DNA synthesis termination is still unclear. Here we report results of functional and structural studies revealing how the human primosome counts RNA-DNA primer length and timely terminates DNA elongation. Using a single-turnover primer extension assay, we defined two factors that determine a mature primer length (∼35-mer): (i) a tight interaction of the C-terminal domain of the DNA primase large subunit (p58C) with the primer 5'-end, and (ii) flexible tethering of p58C and the DNA polymerase alpha catalytic core domain (p180core) to the primosome platform domain by extended linkers. The obtained data allow us to conclude that p58C is a key regulator of all steps of RNA-DNA primer synthesis. The above-described findings provide a notable insight into the mechanism of DNA synthesis termination by a eukaryotic primosome, an important process for ensuring successful primer handover to replication DNA polymerases and for maintaining genome integrity.

Structural and functional insight into mismatch extension by human DNA polymerase α.

Human DNA polymerase α (Polα) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Polα extends the RNA primers generated by primase and provides a springboard for loading other replication factors. Here we provide the structural and functional analysis of the human Polα interaction with a mismatched template:primer. The structure of the human Polα catalytic domain in the complex with an incoming deoxycytidine triphosphate (dCTP) and the template:primer containing a T-C mismatch at the growing primer terminus was solved at a 2.9 Å resolution. It revealed the absence of significant distortions in the active site and in the conformation of the substrates, except the primer 3'-end. The T-C mismatch acquired a planar geometry where both nucleotides moved toward each other by 0.4 Å and 0.7 Å, respectively, and made one hydrogen bond. The binding studies conducted at a physiological salt concentration revealed that Polα has a low affinity to DNA and is not able to discriminate against a mispaired template:primer in the absence of deoxynucleotide triphosphate (dNTP). Strikingly, in the presence of cognate dNTP, Polα showed a more than 10-fold higher selectivity for a correct duplex versus a mismatched one. According to pre-steady-state kinetic studies, human Polα extends the T-C mismatch with a 249-fold lower efficiency due to reduction of the polymerization rate constant by 38-fold and reduced affinity to the incoming nucleotide by 6.6-fold. Thus, a mismatch at the postinsertion site affects all factors important for primer extension: affinity to both substrates and the rate of DNA polymerization.