Telomere C-Strand Fill-In Machinery: New Insights into the Human CST-DNA Polymerase Alpha-Primase Structures and Functions.

Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.

A conserved polar residue plays a critical role in mismatch detection in A-family DNA polymerases.

In A-family DNA polymerases (dPols), a functional 3'-5' exonuclease activity is known to proofread newly synthesized DNA. The identification of a mismatch in substrate DNA leads to transfer of the primer strand from the polymerase active site to the exonuclease active site. To shed more light regarding the mechanism responsible for the detection of mismatches, we have utilized DNA polymerase 1 from Aquifex pyrophilus (ApPol1). The enzyme synthesized DNA with high fidelity and exhibited maximal exonuclease activity with DNA substrates bearing mismatches at the -2 and - 3 positions. The crystal structure of apo-ApPol1 was utilized to generate a computational model of the functional ternary complex of this enzyme. The analysis of the model showed that N332 forms interactions with minor groove atoms of the base pairs at the -2 and - 3 positions. The majority of known A-family dPols show the presence of Asn at a position equivalent to N332. The N332L mutation led to a decrease in the exonuclease activity for representative purine-pyrimidine, and pyrimidine-pyrimidine mismatches at -2 and - 3 positions, respectively. Overall, our findings suggest that conserved polar residues located towards the minor groove may facilitate the detection of position-specific mismatches to enhance the fidelity of DNA synthesis.

Primase and polymerase α tango to make an RNA-DNA hybrid primer.

Several recent cryo-electron microscopy (cryo-EM) studies about the eukaryotic primosome, including the human primosome described by Yin et al. in this issue, have uncovered the structural intricacies between the RNA primase and the DNA polymerase. These studies show that these two partners tango on DNA to synthesize a hybrid primer composed of ~ 10 nucleotide (nt) RNA and ~ 10-nt DNA. They reveal key intermediate steps involved in this process; from the self-inhibited apo state to the initiation of RNA primer synthesis, RNA primer handover to the polymerase, primer elongation by polymerase, and finally, primer termination and release. Remarkably, the polymerase domain orchestrates all major steps during primer synthesis.

A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.

The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.

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.

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.

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.

Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment.

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism. Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5' exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5' exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0-8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0-6.5. The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.

Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1.

Several native and engineered heat-stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72-74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3-5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half-life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high-temperature DNA polymerization.

A non-radioactive DNA synthesis assay demonstrates that elements of the Sigma 1278b Mip1 mitochondrial DNA polymerase domain and C-terminal extension facilitate robust enzyme activity.

The yeast DNA polymerase gamma, Mip1, is a useful tool to investigate the impact of orthologous human disease variants on mitochondrial DNA (mtDNA) replication. However, Mip1 is characterized by a C-terminal extension (CTE) that is not found on orthologous metazoan DNA polymerases, and the CTE is required for robust enzymatic activity. Two MIP1 alleles exist in standard yeast strains, encoding Mip1[S] or Mip1[Σ]. Mip1[S] is associated with reduced mtDNA stability and increased error rates in vivo. Although the Mip1[S] allele was initially identified in S288c, the Mip1[Σ] allele is widely present among available yeast genome sequences, suggesting that it is the wild-type (WT) allele. We developed a novel non-radioactive polymerase gamma assay to assess Mip1 functioning at its intracellular location, the mitochondrial membrane. Membrane fractions were isolated from yeast cells expressing full-length or CTE truncation variants of Mip1[S] or a chimeric Mip1[S] isoform harboring the Mip1[Σ]-specific T661 residue (cMip1 T661). Relative incorporation of digoxigenin (DIG)-11-deoxyuridine monophosphate (DIG-dUMP) by cMip1 T661 was higher than that by Mip1[S]. A cMip1 T661variant lacking 175 C-terminal residues maintained WT levels of DIG-dUMP incorporation, whereas the C-terminal variant lacking 205 residues displayed a significant decrease in incorporation. Newly synthesized DIG-labeled DNA decreased during later phases of reactions carried out at 37°C, suggesting temperature-sensitive destabilization of the polymerase domain and/or increased shuttling of the nascent DNA into the exonuclease domain. Comparative analysis of Mip1 enzyme functions using our novel assay has further demonstrated the importance of the CTE and T661 encoded by MIP1[Σ] in yeast mtDNA replication.

Effect of Molecular Crowding on DNA Polymerase Reactions along Unnatural DNA Templates.

Unnatural nucleic acids are promising materials to expand genetic information beyond the natural bases. During replication, substrate nucleotide incorporation should be strictly controlled for optimal base pairing with template strand bases. Base-pairing interactions occur via hydrogen bonding and base stacking, which could be perturbed by the chemical environment. Although unnatural nucleobases and sugar moieties have undergone extensive structural improvement for intended polymerization, the chemical environmental effect on the reaction is less understood. In this study, we investigated how molecular crowding could affect native DNA polymerization along various templates comprising unnatural nucleobases and sugars. Under non-crowding conditions, the preferred incorporation efficiency of pyrimidine deoxynucleotide triphosphates (dNTPs) by the Klenow fragment (KF) was generally high with low fidelity, whereas that of purine dNTPs was the opposite. However, under crowding conditions, the efficiency remained almost unchanged with varying preferences in each case. These results suggest that hydrogen bonding and base-stacking interactions could be perturbed by crowding conditions in the bulk solution and polymerase active center during transient base pairing before polymerization. This study highlights that unintended dNTP incorporation against unnatural nucleosides could be differentiated in cases of intracellular reactions.

Elucidating the Inhibitory Effect of Resveratrol and Its Structural Analogs on Selected Nucleotide-Related Enzymes.

Resveratrol, the most widely studied natural phytochemical, has been shown to interact with different target proteins. Previous studies show that resveratrol binds and inhibits DNA polymerases and some other enzymes; however, the binding and functioning mechanisms remain unknown. The elucidated knowledge of inhibitory mechanisms of resveratrol will assist us in new drug discovery. We utilized molecular docking and molecular dynamics (MD) simulation to reveal how resveratrol and structurally similar compounds bind to various nucleotide-dependent enzymes, specifically, DNA polymerases, HIV-1 reverse transcriptase, and ribonucleotide reductase. The results show that resveratrol and its analogs exert their inhibitory effects by competing with the substrate dNTPs in these enzymes and blocking elongation of chain polymerization. In addition, the results imply that resveratrol binds to a variety of other ATP-/NTP-binding proteins.

Display of functional nucleic acid polymerase on Escherichia coli surface and its application in directed polymerase evolution.

We report a first of its kind functional cell surface display of nucleic acid polymerase and its directed evolution to efficiently incorporate 2'-O-methyl nucleotide triphosphates (2'-OMe-NTPs). In the development of polymerase cell surface display, two autotransporter proteins (Escherichia coli adhesin involved in diffuse adherence and Pseudomonas aeruginosa esterase A [EstA]) were employed to transport and anchor the 68-kDa Klenow fragment (KF) of E. coli DNA polymerase I on the surface of E. coli. The localization and function of the displayed KF were verified by analysis of cell outer membrane fractions, immunostaining, and fluorometric detection of synthesized DNA products. The EstA cell surface display system was applied to evolve KF for the incorporation of 2'-OMe-NTPs and a KF variant with a 50.7-fold increased ability to successively incorporate 2'-OMe-NTPs was discovered. Expanding the scope of cell-surface displayable proteins to the realm of polymerases provides a novel screening tool for tailoring polymerases to diverse application demands in a polymerase chain reaction and sequencing-based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which the heat-lysis step is bypassed and thus allows the screening of mesophilic polymerases with broad application potentials ranging from diagnostics and DNA sequencing to replication of synthetic genetic polymers.

E. coli DNA Polymerase I and the Klenow Fragment.

Escherichia coli DNA Pol I can carry out three enzymatic reactions: It possesses 5' → 3' DNA polymerase activity and 3' → 5' and 5' → 3' exonuclease activity. Pol I can be cleaved by mild treatment with subtilisin into two fragments; the larger fragment is known as the Klenow fragment. The Klenow fragment retains the polymerizing activity and the 3' → 5' exonuclease of the holo-enzyme but lacks its powerful 5' → 3' exonuclease activity. These enzymes and their applications in molecular cloning are introduced here.

Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase ß-DNA complexes.

Eukaryotic DNA polymerase ß (Pol ß) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol ß has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol ß. First, using a doubly labeled DNA construct, we show that Pol ß bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol ß and donor-labeled DNA, we visualized dynamic fingers closing in single Pol ß-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol ß, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol ß reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

Enzymatic Cleavage of 3'-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis.

The reversible dye-terminator (RDT)-based DNA sequencing-by-synthesis (SBS) chemistry has driven the advancement of the next-generation sequencing technologies for the past two decades. The RDT-based SBS chemistry relies on the DNA polymerase reaction to incorporate the RDT nucleotide (NT) for extracting DNA sequence information. The main drawback of this chemistry is the "DNA scar" issue since the removal of dye molecule from the RDT-NT after each sequencing reaction cycle leaves an extra chemical residue in the newly synthesized DNA. To circumvent this problem, we designed a novel class of reversible (2-aminoethoxy)-3-propionyl (Aep)-dNTPs by esterifying the 3'-hydroxyl group (3'-OH) of deoxyribonucleoside triphosphate (dNTP) and examined the NT-incorporation activities by A-family DNA polymerases. Using the large fragment of both Bacillus stearothermophilus (BF) and E. coli DNA polymerase I (KF) as model enzymes, we further showed that both proteins efficiently and faithfully incorporated the 3'-Aep-dNMP. Additionally, we analyzed the post-incorporation product of N + 1 primer and confirmed that the 3'-protecting group of 3'-Aep-dNMP was converted back to a normal 3'-OH after it was incorporated into the growing DNA chain by BF. By applying all four 3'-Aep-dNTPs and BF for an in vitro DNA synthesis reaction, we demonstrated that the enzyme-mediated deprotection of inserted 3'-Aep-dNMP permits a long, continuous, and scar-free DNA synthesis.

Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO-POL enzyme.

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain-especially the O helix-to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO-POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy.

Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas' DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas' DNA. Modified nucleotides are incorporated into mycoplasmas' DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes.