Substrates For DNA Polymerases Research Articles

Bacteriophage-related epigenetic natural and non-natural pyrimidine nucleotides and their influence on transcription with T7 RNA polymerase

DNA modifications on pyrimidine nucleobases play diverse roles in biology such as protection of bacteriophage DNA from enzymatic cleavage, however, their role in the regulation of transcription is underexplored. We have designed and synthesized a series of uracil 2ʹ-deoxyribonucleosides and 5ʹ-O-triphosphates (dNTPs) bearing diverse modifications at position 5 of nucleobase, including natural nucleotides occurring in bacteriophages, α-putrescinylthymine, α-glutaminylthymine, 5-dihydroxypentyluracil, and methylated or non-methylated 5-aminomethyluracil, and non-natural 5-sulfanylmethyl- and 5-cyanomethyluracil. The dNTPs bearing basic substituents were moderate to poor substrates for DNA polymerases, but still useful in primer extension synthesis of modified DNA. Together with previously reported epigenetic pyrimidine nucleotides, they were used for the synthesis of diverse DNA templates containing a T7 promoter modified in the sense, antisense or in both strands. A systematic study of the in vitro transcription with T7 RNA polymerase showed a moderate positive effect of most of the uracil modifications in the non-template strand and some either positive or negative influence of modifications in the template strand. The most interesting modification was the non-natural 5-cyanomethyluracil which showed significant positive effect in transcription.

Optimized Method for the Synthesis of Alkyne-Modified 2'-Deoxynucleoside Triphosphates.

A general approach is presented for synthesizing alkyne-modified nucleoside triphosphates via the Sonogashira cross-coupling reaction of unprotected halogenated 2'-deoxynucleoside, followed by monophosphorylation and the reaction of the corresponding phosphoromorpholidate with tributylammonium pyrophosphate. A highly efficient approach for the milligram-scale synthesis of base-modified nucleoside triphosphates with an amino acid-like side chain was developed. The present chemical method outweighs the other reported methods of a base-modified nucleoside triphosphates synthesis in terms of it being a protection-free strategy, the shortening of reaction steps, and increased yields (about 70%). The resulting 8-alkynylated dATP was tested as a substrate for DNA polymerases in a primer extension reaction.

Glyco-DNA: Enzymatic Synthesis of Base-Modified and Hypermodified DNA Displaying up to Four Different Monosaccharide Units in the Major Groove.

A portfolio of six modified 2'-deoxyribonucleoside triphosphate (dNTP) derivatives derived from 5-substituted pyrimidine or 7-substituted 7-deazapurine bearing different carbohydrate units (d-glucose, d-galactose, d-mannose, l-fucose, sialic acid and N-Ac-d-galactosamine) tethered through propargyl-glycoside linker was designed and synthesized via the Sonogashira reactions of halogenated dNTPs with the corresponding propargyl-glycosides. The nucleotides were found to be good substrates for DNA polymerases in enzymatic primer extension and PCR synthesis of modified and hypermodified DNA displaying up to four different sugars. Proof of concept binding study of sugar-modified oligonucleotides with concanavalin A showed positive effect of avidity and sugar units count.

Trans-Cyclooctene- and Bicyclononyne-Linked Nucleotides for Click Modification of DNA with Fluorogenic Tetrazines and Live Cell Metabolic Labeling and Imaging.

A series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 2- or 4-linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer were designed and synthesized. They were found to be good substrates for KOD XL DNA polymerase for primer extension enzymatic synthesis of modified oligonucleotides. We systematically tested and compared the reactivity of TCO- and BCN-modified nucleotides and DNA with several fluorophore-containing tetrazines in inverse electron-demand Diels-Alder (IEDDA) click reactions to show that the longer linker is crucial for efficient labeling. The modified dNTPs were transported into live cells using the synthetic transporter SNTT1, incubated for 1 h, and then treated with tetrazine conjugates. The PEG3-linked 4TCO and BCN nucleotides showed efficient incorporation into genomic DNA and good reactivity in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells within time periods as short as 15 min. The BCN-linked nucleotide in combination with TAMRA-linked (TAMRA = carboxytetramethylrhodamine) tetrazine was also efficiently used for staining of DNA for flow cytometry. This methodology is a new approach for in cellulo metabolic labeling and imaging of DNA synthesis which is shorter, operationally simple, and overcomes several problems of previously used methods.

Nucleotides Bearing Red Viscosity-Sensitive Dimethoxy-Bodipy Fluorophore for Enzymatic Incorporation and DNA Labeling.

Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 3,3'-dimethoxy-2,2'-diphenyl-6-(4-hydroxyphenyl)-bodipy fluorophore attached through a propargyl or propargyl-triethylene glycol linker to position 5 of 2'-deoxycytidine were designed and synthesized. They exerted bright red fluorescence and good sensitivity to viscosity changing their lifetime from 1.6 to 4.5 ns. The modifed dNTPs were substrates for DNA polymerases and were used in enzymatic synthesis of labeled DNA through primer extension. The modified DNA probes served as viscosity sensors responding to protein binding by changes of lifetime. The nucleotide with longer linker (dCpegMOBTP) was transported to live cells and incorporated into the genomic DNA, which can be useful for staining of DNA and imaging of DNA synthesis.

Synthesis of Cy5-Labelled C5-Alkynyl-modified cytidine triphosphates via Sonogashira coupling for DNA labelling

New applications of palladium-catalyzed Sonogashira-type cross-coupling reaction between C5-halogenated 2′-deoxycytidine-5′-monophosphate and novel cyanine dyes with a terminal alkyne group have been developed. The present methodology allows to synthesize of fluorescently labeled C5-nucleoside triphosphates with different acetylene linkers between the fluorophore and pyrimidine base in good to excellent yields under mild reaction conditions. Modified 2′-deoxycytidine-5′-triphosphates were shown to be good substrates for DNA polymerases and were incorporated into the DNA by polymerase chain reaction.

Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression.

Apurinic/apyrimidinic endonuclease 1/redox effector-1 (Ape1/Ref-1) is the major apurinic/apyrimidinic (AP) endonuclease in mammalian cells. It functions mainly in the base excision repair pathway to create a suitable substrate for DNA polymerases. Human Ape1 protein can activate some transcription factors to varying degrees, dependent on its N-terminal, unstructured domain, and some of the cysteines within it, apparently via a redox mechanism in some cases. Many cancer studies also suggest that Ape1 has potential for prognosis in terms of the protein level or intracellular localization. While homozygous disruption of the Ape1 structural gene APEX1 in mice causes embryonic lethality, and most studies in cell culture indicate that the expression of Ape1 is essential, some recent studies reported the isolation of viable APEX1 knockout cells with only mild phenotypes. It has not been established by what mechanism the Ape1-null cell lines cope with the endogenous DNA damage that the enzyme normally handles. We review the enzymatic and other activities of Ape1 and the recent studies of the properties of the APEX1 knockout lines. The APEX1 deletions in CH12F3 and HEK293 FT provide an opportunity to test for possible off-target effects of Ape1 inhibition. For this work, we tested the Ape1 endonuclease inhibitor Compound 3 and the redox inhibitor APX2009. Our results confirmed that both APEX1 knockout cell lines are modestly more sensitive to killing by an alkylating agent than their Ape1-proficient cells. Surprisingly, the knockout lines showed equal sensitivity to direct killing by either inhibitor, despite the lack of the target protein. Moreover, the CH12F3 APEX1 knockout was even more sensitive to Compound 3 than its APEX1+ counterpart. Thus, it appears that both Compound 3 and APX2009 have off-target effects. In cases where this issue may be important, it is advisable that more specific endpoints than cell survival be tested for establishing mechanism.

Epigenetic Pyrimidine Nucleotides in Competition with Natural dNTPs as Substrates for Diverse DNA Polymerases.

Five 2′-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2′-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.

Microenvironment-Sensitive Fluorescent Nucleotide Probes from Benzofuran, Benzothiophene, and Selenophene as Substrates for DNA Polymerases.

DNA polymerases can process a wide variety of structurally diverse nucleotide substrates, but the molecular basis by which the analogs are processed is not completely understood. Here, we demonstrate the utility of environment-sensitive heterocycle-modified fluorescent nucleotide substrates in probing the incorporation mechanism of DNA polymerases in real time and at the atomic level. The nucleotide analogs containing a selenophene, benzofuran, or benzothiophene moiety at the C5 position of 2'-deoxyuridine are incorporated into oligonucleotides (ONs) with varying efficiency, which depends on the size of the heterocycle modification and the DNA polymerase sequence family used. KlenTaq (A family DNA polymerase) is sensitive to the size of the modification as it incorporates only one heterobicycle-modified nucleotide into the growing polymer, whereas it efficiently incorporates the selenophene-modified nucleotide analog at multiple positions. Notably, in the single nucleotide incorporation assay, irrespective of the heterocycle size, it exclusively adds a single nucleotide at the 3'-end of a primer, which enabled devising a simple two-step site-specific ON labeling technique. KOD and Vent(exo-) DNA polymerases, belonging to the B family, tolerate all the three modified nucleotides and produce ONs with multiple labels. Importantly, the benzofuran-modified nucleotide (BFdUTP) serves as an excellent reporter by providing real-time fluorescence readouts to monitor enzyme activity and estimate the binding events in the catalytic cycle. Further, a direct comparison of the incorporation profiles, fluorescence data, and crystal structure of a ternary complex of KlenTaq DNA polymerase with BFdUTP poised for catalysis provides a detailed understanding of the mechanism of incorporation of heterocycle-modified nucleotides.

Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites

DNA interstrand cross-links (ICLs) are extremely deleterious DNA lesions, which can block different DNA transactions. A major step in ICL repair involves strand cleavage activities flanking the cross-linking site, also known as unhooking. The cleavage generates a single-stranded DNA remnant attached to the unbroken strand, often referred to as the unhooked ICL repair intermediates. The unhooked ICLs are substrates for specialized DNA polymerases, leading to the eventual restoration of the duplex DNA structure. Although these repair events have been outlined, the understanding of molecular details of the repair pathways has been hindered by the difficulty of preparing structurally defined ICL repair intermediates. Here, we present a straightforward method to prepare model ICL repair intermediates derived from a ubiquitous type of endogenous DNA modification, abasic (AP) sites. AP-derived ICLs have emerged as an important type of endogenous ICLs. We developed the method based on commercially available materials without the requirement of synthetic chemistry expertise. The method is expected to be accessible to any interested labs in the DNA repair community.• The method exploits the alkaline lability of ribonucleotides and uses designer oligonucleotides to create ICL repair intermediates with varying lengths of the unhooked strand.• Strand cleavage at ribonucleotides is achieved using NaOH, which avoids the potential for incomplete digestion during enzymatic workup due to specific substrate structures.• The method is grounded on the high cross-linking yield between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination, developed by Gates and colleagues.

Nucleoside boranephosphonates as potential therapeutic agents

Nucleoside boranephosphonates are nucleotide analogues in which one of the non-bridging oxygen atom of the phosphate part has been replaced by a borane group (-BH3). This modification imparts a wide spectrum of biological activity, e.g., activation of ribonuclease H, resistance to endo- and exonucleases, and their respective triphosphates are good substrates for DNA and RNA polymerases. Nucleoside boranephosphonate derivatives are used in antisense therapy, silencing gene expression using siRNA strategies, and as potential antiviral and anti-cancer prodrugs. Boranephosphonates find also applications as aptamers and as substrates in a new method of DNA sequencing. This review briefly presents potential biological applications of nucleoside boranephosphonates.

Polymerase Synthesis of DNA Containing Iodinated Pyrimidine or 7‐Deazapurine Nucleobases and Their Post‐synthetic Modifications through the Suzuki‐Miyaura Cross‐Coupling Reactions

All four iodinated 2'-deoxyribonucleoside triphosphates (dNTPs) derived from 5-iodouracil, 5-iodocytosine, 7-iodo-7-deazaadenine and 7-iodo-7-deazaguanine were prepared and studied as substrates for KOD XL DNA polymerase. All of the nucleotides were readily incorporated by primer extension and by PCR amplification to form DNA containing iodinated nucleobases. Systematic study of the Suzuki-Miyaura cross-coupling reactions with two bulkier arylboronic acids revealed that the 5-iodopyrimidines were more reactive and gave cross-coupling products both in the terminal or internal position in single-stranded oligonucleotides (ssONs) and in the terminal position of double-stranded DNA (dsDNA), whereas the 7-iodo-7-deazapurines were less reactive and gave cross-coupling products only in the terminal position. None of the four iodinated bases reacted in an internal position of dsDNA. These findings are useful for the use of the iodinated nucleobases for post-synthetic modification of DNA with functional groups for various applications.

1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking with Arginine-Containing Peptides and Proteins.

Linear or branched 1,3‐diketone‐linked thymidine 5′‐O‐mono‐ and triphosphate were synthesized through CuAAC click reaction of diketone‐alkynes with 5‐azidomethyl‐dUMP or ‐dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5‐dioxohexyl group (HDO) efficiently reacted with arginine‐containing peptides to form stable pyrimidine‐linked conjugates, whereas the branched 2‐acetyl‐3‐oxo‐butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross‐linking with Arg‐containing peptides or proteins (e.g. histones).

1,3‐Diketone‐Modified Nucleotides and DNA for Cross‐Linking with Arginine‐Containing Peptides and Proteins

AbstractLinear or branched 1,3‐diketone‐linked thymidine 5′‐O‐mono‐ and triphosphate were synthesized through CuAAC click reaction of diketone‐alkynes with 5‐azidomethyl‐dUMP or ‐dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5‐dioxohexyl group (HDO) efficiently reacted with arginine‐containing peptides to form stable pyrimidine‐linked conjugates, whereas the branched 2‐acetyl‐3‐oxo‐butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross‐linking with Arg‐containing peptides or proteins (e.g. histones).

No Association between Relapse Risk and Thiopurine Methyltransferase Activity By Either Geno- or Phenotype in the NOPHO ALL-2008 Protocol

Background6-mercaptopurine/methotrexate (6MP/MTX) maintenance therapy is essential for high cure rates of childhood acute lymphoblastic leukemia (ALL). 6MP exerts its cytotoxicity through conversion into thioguanine nucleotides (TGN) that are substrates for DNA polymerase and compete with normal guanine for DNA incorporation. DNA-TGN occasionally mismatches to thymidine causing cell death due to futile attempts of repetitive mismatch repair. We recently showed that higher levels of DNA-incorporated TGN were associated with a reduced relapse risk in non-high risk ALL (Nielsen, Lancet Oncol 2017). Common germline variants in TPMT that codes for thiopurine methyltransferase (TPMT) lead to reduced TPMT activity and higher levels of cytosol TGN when measured in erythrocytes. Thus, low TPMT activity could theoretically lead to higher DNA-TGN and a lower relapse rate. Low TPMT activity (phenotype) has been associated with a lower relapse risk in the NOPHO ALL92 protocol (Schmiegelow, Leukemia 2009). We explored if TPMT status evaluated by either geno- or phenotype was related to DNA-TGN levels and relapse rate in the NOPHO ALL2008 protocol.MethodTPMTgenotype and repeated phenotyping (2141 measurements in total), and levels of DNA-TGN (10830 measurements in total) during maintenance therapy were determined for 918 children treated for non-high risk ALL (89% of all eligible patients). On a subset (N=204) TPMT phenotype was available both at diagnosis and during maintenance therapy. 6MP was started at 50 and 75 mg/m2 for TPMT heterozygous and wildtype patients, respectively, and subsequently adjusted to a target WBC of 1.5-3.0 x109/L. Cox analyses were adjusted by age, sex and WBC at diagnosis.Results78 of the 918 non-high risk patients were TPMT heterozygous (none homozygous deficient). TPMT activities were significantly higher during maintenance therapy than at diagnosis for both TPMT wildtype and heterozygous children (N=204; wildtype: 16.6 vs. 11.3 U/mL ery., p <0.001; heterozygous: 9.9 vs. 6.8 U/mL ery., p=0.047). Mean DNA-TGN levels were higher for TPMT heterozygous than wildtype children (N=523, children in remission at end of therapy with Maintenance II measurements, median 760.9 fmol/µg, range 125.7−1255.0 vs. 494.9, range 44.0−1559.0, p <0.001; Figure). The 5-year cumulative incidence of relapse was 4.2% (95% CI: 0-8.7%) for TPMT heterozygous and 5.7% (95% CI: 3.8-7.5%) forwildtype children (hazard ratio =0.87, 95% CI: 0.27−2.83, p=0.81). No association between mean TPMT activity during maintenance therapy (as a time-dependent covariate) and relapse-specific hazard was seen (N=812; hazard ratio =0.98 per one unit increase in TPMT activity (U/mL ery.), 95% CI: 0.89−1.08, p=0.63). In patients with positive MRD at end of induction (d29) results were similar (N=476; hazard ratio =1.04 per one unit increase in TPMT activity (U/mL ery.), 95% CI: 0.93−1.16, p=0.50).ConclusionAlthough mean DNA-TGN levels were higher in TPMT heterozygous than wildtype children, variability was high in both groups and DNA-TGN levels were largely overlapping, and no association with relapse hazard was seen for neither TPMT geno- nor phenotype. In accordance with previously published data TPMT phenotype determinations done in erythrocytes present at ALL diagnosis can lead to TPMT misclassification.FIGUREa) Mean DNA-TGN levels (N=523) during Maintenance II of children in clinical remission with completed therapy and at least one measurement.b) Cumulative incidence curves of relapse of children carrying TPMT wildtype vs. heterozygous. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing.

We report a series of 2'-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11]1-), [3,3'-iron-bis(1,2-dicarbollide)]- (FESAN, [Fe(C2B9H11)2]2-) or [3,3'-cobalt-bis(1,2-dicarbollide)]- (COSAN, [Co(C2B9H11)2]2-) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dNXTPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

Synthesis of Polyanionic C5-Modified 2'-Deoxyuridine and 2'-Deoxycytidine-5'-Triphosphates and Their Properties as Substrates for DNA Polymerases.

Modified 2′-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.

SiRNAs containing 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMCNTPs.

We recently reported the synthesis of 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2′-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2′-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5′ phosphate, suggesting that the 2′-F-NMC is a poor substrate for 5′ kinases. In mice, the 2′-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2′-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5′-phosphate mimic 5′-(E)-vinylphosphonate was attached to the 2′-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2′-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2′-F-NMC. Finally, the 5′-triphosphate of 2′-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

Mechanism of Deoxyguanosine Diphosphate Insertion by Human DNA Polymerase β.

DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (kpol), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme-substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates.

Membrane Permeable, Bioreversibly Modified Prodrugs of Nucleoside Diphosphate-γ-Phosphonates

Nucleoside reverse transcriptase inhibitors (NRTIs) are widely used as antiviral and anticancer agents, although they require intracellular phosphorylation into their antivirally active form, the triphosphorylated nucleoside analogue metabolites. We report on the synthesis and characterization of a new class of nucleoside triphosphate analogues comprising a C-alkyl-phosphonate moiety replacing the γ-phosphate. These compounds were converted into bioreversibly modified lipophilic prodrugs at the γ-phosphonate by the attachment of an acyloxybenzyl (ester) or an alkoxycarbonyloxybenzyl (carbonate) group. Such compounds formed γ-C-(alkyl)-nucleoside triphosphate analogues with high selectivity because of an enzyme-triggered delivery mechanism. The latter compounds were very stable in CD4+ T-lymphocyte (CEM cell) extracts, and they were substrates for HIV-reverse transcriptase without being substrates for DNA-polymerases α, β, and γ. In antiviral assays, the excellent antiviral activity of the prodrugs that was found in CEM/0 cells was completely kept in CEM/TK- cells. The activity was improved by 3 logs as compared to the parent nucleoside d4T.