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The FEBS Journal Sep 2014The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation... (Review)
Review
The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation effects on bases within the DNA structure. Several enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a number of representatives in clear structural and kinetic detail. In this review, we focus in detail on those examples where clear evidence has been produced using high-resolution structural studies. Comparing the protein fold and architecture of the enzyme active sites, two main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes associated with attack at the α-phosphorus, it is shown that coordination of the γ-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the β-phosphorus atom mainly coordinate the α- and the β-phosphate only. Characteristic differences are also observed with respect to the role of the metal ion cofactor (Mg(2+) ) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution.
Topics: Amino Acid Sequence; Animals; Catalytic Domain; Consensus Sequence; DNA Repair; DNA Repair Enzymes; Deoxyribonucleotides; Humans; Models, Molecular; Molecular Sequence Data; Phosphoric Monoester Hydrolases
PubMed: 25052017
DOI: 10.1111/febs.12941 -
Molecules (Basel, Switzerland) Nov 2012Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate... (Review)
Review
Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate analogues represents a versatile platform for the facile and mild generation of (highly) functionalized nucleic acids. Numerous modified triphosphates have been utilized in a broad palette of applications spanning from DNA-tagging and -labeling to the generation of catalytic nucleic acids. This review will focus on the recent progress made in the synthesis of modified nucleoside triphosphates as well as on the understanding of the mechanisms underlying their polymerase acceptance. In addition, the usefulness of chemically altered dNTPs in SELEX and related methods of in vitro selection will be highlighted, with a particular emphasis on the generation of modified DNA enzymes (DNAzymes) and DNA-based aptamers.
Topics: Base Sequence; DNA; DNA Adducts; DNA, Catalytic; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Nucleotides; Protein Binding; SELEX Aptamer Technique
PubMed: 23154273
DOI: 10.3390/molecules171113569 -
Scientific Reports Jan 2020The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and...
The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 10 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.
Topics: Click Chemistry; Copper; Cycloaddition Reaction; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Deoxyuracil Nucleotides; HCT116 Cells; HEK293 Cells; Humans; K562 Cells; Rhodamines; Staining and Labeling; Thymine Nucleotides
PubMed: 31953472
DOI: 10.1038/s41598-020-57463-3 -
Journal of the American Chemical Society Jul 2019Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base...
Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.
Topics: Base Pairing; Deoxyribonucleotides; Genetic Code; Nucleotides; Protein Biosynthesis; Synthetic Biology; Transcription, Genetic
PubMed: 31241334
DOI: 10.1021/jacs.9b02075 -
Nucleic Acids Research Apr 2019For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro...
For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.
Topics: Acetylgalactosamine; Animals; Deoxyribonucleotides; Female; Fluorine; Humans; Male; RNA, Small Interfering; Rats; Rats, Sprague-Dawley
PubMed: 30820542
DOI: 10.1093/nar/gkz140 -
Science Advances Jun 2021Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template...
Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Å crystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2'-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair.
Topics: Animals; DNA; DNA Repair; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Humans; Mammals; RNA; Ribonucleotides
PubMed: 34117057
DOI: 10.1126/sciadv.abf1771 -
Nucleic Acids Research Sep 2019A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read...
A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.
Topics: DNA-Directed DNA Polymerase; Deoxyribonucleosides; Deoxyribonucleotides; High-Throughput Nucleotide Sequencing; Limit of Detection; Microscopy, Fluorescence; Oligodeoxyribonucleotides; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 31318971
DOI: 10.1093/nar/gkz611 -
Nucleic Acids Research Apr 2016Experimental characterization of the structural couplings in free B-DNA in solution has been elusive, because of subtle effects that are challenging to tackle. Here, the...
Experimental characterization of the structural couplings in free B-DNA in solution has been elusive, because of subtle effects that are challenging to tackle. Here, the exploitation of the NMR measurements collected on four dodecamers containing a substantial set of dinucleotide sequences provides new, consistent correlations revealing the DNA intrinsic mechanics. The difference between two successive residual dipolar couplings (ΔRDCs) involving C6/8-H6/8, C3'-H3' and C4'-H4' vectors are correlated to the(31)P chemical shifts (δP), which reflect the populations of the BI and BII backbone states. The δPs are also correlated to the internucleotide distances (Dinter) involving H6/8, H2' and H2″ protons. Calculations of NMR quantities on high resolution X-ray structures and controlled models of DNA enable to interpret these couplings: the studied ΔRDCs depend mostly on roll, while Dinterare mainly sensitive to twist or slide. Overall, these relations demonstrate how δP measurements inform on key inter base parameters, in addition to probe the BI↔BII backbone equilibrium, and shed new light into coordinated motions of phosphate groups and bases in free B-DNA in solution. Inspection of the 5' and 3' ends of the dodecamers also supplies new information on the fraying events, otherwise neglected.
Topics: DNA, B-Form; Deoxyribonucleotides; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation
PubMed: 26883628
DOI: 10.1093/nar/gkw084 -
Nucleic Acids Research Nov 2023The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases...
The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2'-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.
Topics: Catalytic Domain; Crystallography, X-Ray; Deoxyribonucleotides; DNA; DNA Polymerase II; Ribonucleotides; Saccharomyces cerevisiae
PubMed: 37819038
DOI: 10.1093/nar/gkad817 -
Analytical Chemistry Nov 2011The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned...
The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.
Topics: Base Sequence; Candida; DNA, Fungal; Deoxyribonucleotides; Electrodes; Enzymes; Microfluidic Analytical Techniques; Sequence Analysis, DNA; Templates, Genetic
PubMed: 21932784
DOI: 10.1021/ac201416j