Development of a 'clickable' non-natural nucleotide to visualize the replication of non-instructional DNA lesions

The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using 'click' chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.     

Rational attempts to optimize non-natural nucleotides for selective incorporation opposite an abasic site

Translesion DNA synthesis represents the ability of a DNA polymerase to misinsert a nucleotide opposite a DNA lesion. Previous kinetic studies of the bacteriophage T4 DNA polymerase using a series of non-natural nucleotides suggest that pi-electron density of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site, a nontemplating DNA lesion. However, it is surprising that these nonhydrogen-bonding analogues can also be incorporated opposite natural templating DNA with variable degrees of efficiency. In this article, we describe attempts to achieve selectivity for incorporation opposite the abasic site through optimization of pi-electron density and shape of the nucleobase. Toward this goal, we report the synthesis and enzymatic characterization of two novel nucleotide analogues, 5-napthyl-indolyl-2'-deoxyriboside triphosphate (5-NapITP) and 5-anthracene-indolyl-2'-deoxyriboside triphosphate (5-AnITP). The overall catalytic efficiency for their incorporation opposite an abasic site is similar to that of other non-natural nucleotides such as 5-NITP and 5-PhITP that contain significant pi-electron density. As expected, the incorporation of either 5-NapITP or 5-AnITP opposite templating DNA is reduced and presumably reflects steric constraints imposed by the large size of the polycyclic aromatic moieties. Furthermore, 5-NapITP is a chain terminator of translesion DNA synthesis because the DNA polymerase is unable to extend beyond the incorporated non-natural nucleotide. In addition, idle turnover measurements confirm that 5-NapIMP is stably incorporated opposite damaged DNA, and this enhancement reflects the overall favorable incorporation kinetic parameters coupled with a reduction in excision by the exonuclease-proofreading activity of the enzyme. On the basis of these data, we provide a comprehensive assessment of the potential role of pi-electron surface area for nucleotide incorporation opposite templating and nontemplating DNA catalyzed by the bacteriophage T4 DNA polymerase.     

Evaluating the contribution of base stacking during translesion DNA replication

Despite the nontemplating nature of the abasic site, dAMP is often preferentially inserted opposite the lesion, a phenomenon commonly referred to as the "A-rule". We have evaluated the molecular mechanism accounting for this unique behavior using a thorough kinetic approach to evaluate polymerization efficiency during translesion DNA replication. Using the bacteriophage T4 DNA polymerase, we have measured the insertion of a series of modified nucleotides and have demonstrated that increasing the size of the nucleobase does not correlate with increased insertion efficiency opposite an abasic site. One analogue, 5-nitroindolyl-2'-deoxyriboside triphosphate, was unique as it was inserted opposite the lesion with approximately 1000-fold greater efficiency compared to that for dAMP insertion. Pre-steady-state kinetic measurements yield a kpol value of 126 s(-1) and a Kd value of 18 microM for the insertion of 5-nitroindolyl-2'-deoxyriboside triphosphate opposite the abasic site. These values rival those associated with the enzymatic formation of a natural Watson-Crick base pair. These results not only reiterate that hydrogen bonding is not necessary for nucleotide insertion but also indicate that the base-stacking and/or desolvation capabilities of the incoming nucleobase may indeed play the predominant role in generating efficient DNA polymerization. A model accounting for the increase in catalytic efficiency of this unique nucleobase is provided and invokes pi-pi stacking interactions of the aromatic moiety of the incoming nucleobase with aromatic amino acids present in the polymerase's active site. Finally, differences in the rate of 5-nitroindolyl-2'-deoxyriboside triphosphate insertion opposite an abasic site are measured between the bacteriophage T4 DNA polymerase and the Klenow fragment. These kinetic differences are interpreted with regard to the differences in various structural components between the two enzymes and are consistent with the proposed model for DNA polymerization.     

Caught bending the A-rule: crystal structures of translesion DNA synthesis with a non-natural nucleotide

Damage to DNA involving excision of the nucleobase at the N-glycosidic bond forms abasic sites. If a nucleotide becomes incorporated opposite an unrepaired abasic site during DNA synthesis, most B family polymerases obey the A-rule and preferentially incorporate dAMP without instruction from the template. In addition to being potentially mutagenic, abasic sites provide strong blocks to DNA synthesis. A previous crystal structure of an exonuclease deficient variant of the replicative B family DNA polymerase from bacteriophage RB69 (RB69 gp43 exo-) illustrated these properties, showing that the polymerase failed to translocate the DNA following insertion of dAMP opposite an abasic site. We examine four new structures depicting several steps of translesion DNA synthesis by RB69 gp43 exo-, employing a non-natural purine triphosphate analogue, 5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is incorporated more efficiently than dAMP opposite abasic sites. Our structures indicate that a dipole-induced dipole stacking interaction between the 5-nitro group and base 3' to the templating lesion explains the enhanced kinetics of 5-NITP. As with dAMP, the DNA fails to translocate following insertion of 5-NIMP, although distortions at the nascent primer terminus contribute less than previously thought in inducing the stall, given that 5-NIMP preserves relatively undistorted geometry at the insertion site following phosphoryl transfer. An open ternary configuration, novel in B family polymerases, reveals an initial template independent binding of 5-NITP adjacent to the active site of the open polymerase, suggesting that closure of the fingers domain shuttles the nucleotide to the active site while testing the substrate against the template.     

The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an "ajar" intermediate conformation in the nucleotide selection mechanism

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established "open" and "closed" states. In this "ajar" conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.     

Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis.     

Mechanism and dynamics of translesion DNA synthesis catalyzed by the Escherichia coli Klenow fragment

Translesion DNA synthesis represents the ability of a DNA polymerase to incorporate and extend beyond damaged DNA. In this report, the mechanism and dynamics by which the Escherichia coli Klenow fragment performs translesion DNA synthesis during the misreplication of an abasic site were investigated using a series of natural and non-natural nucleotides. Like most other high-fidelity DNA polymerases, the Klenow fragment follows the "A-rule" of translesion DNA synthesis by preferentially incorporating dATP opposite the noninstructional lesion. However, several 5-substituted indolyl nucleotides lacking classical hydrogen-bonding groups are incorporated approximately 100-fold more efficiently than the natural nucleotide. In general, analogues that contain large substituent groups in conjunction with significant pi-electron density display the highest catalytic efficiencies ( k cat/ K m) for incorporation. While the measured K m values depend upon the size and pi-electron density of the incoming nucleotide, k cat values are surprisingly independent of both biophysical features. As expected, the efficiency by which these non-natural nucleotides are incorporated opposite templating nucleobases is significantly reduced. This reduction reflects minimal increases in K m values coupled with large decreases in k cat values. The kinetic data obtained with the Klenow fragment are compared to that of the high-fidelity bacteriophage T4 DNA polymerase and reveal distinct differences in the dynamics by which these non-natural nucleotides are incorporated opposite an abasic site. These biophysical differences argue against a unified mechanism of translesion DNA synthesis and suggest that polymerases employ different catalytic strategies during the misreplication of damaged DNA.     

Detection of terminal deoxynucleotidyl transferase (TdT) by flow cytometry in leukemic disorders

We applied a new technique to the detection of intracellular TdT in 26 leukemic patients, including 16 non-T acute lymphoblastic leukemia (ALL), four T-ALL, one T-lymphoblastic lymphoma in leukemia phase, one undifferentiated leukemia, one de novo lymphoblastic phase of chronic myeloid leukemia, and three acute monocytic leukemias (AMOL). Mononuclear cell suspensions were incubated in saponin to permeabilize the cell membrane. The cells were then stained by indirect immunofluorescence (IF) using anti-human TdT monoclonal antibodies and were analyzed by flow cytometry (FCM). The TdT results were compared with those obtained by biochemical TdT assay (26 cases), immunoperoxidase determination (PAP) (12 cases), and fluorescence microscopy (seven cases). The results obtained by PAP and fluorescence microscopy were 100% concordant with those obtained by FCM and biochemical assay. TdT determination by FCM allows the analysis of large numbers of cells in a fast, objective, and reliable manner, as compared with biochemical assay, PAP, and fluorescence microscopy determinations.     

Stimulation of protocollagen proline hydroxylase activity by nucleoside triphosphates.

Activity of purified protocollagen proline hydroxylase was enhanced several fold by addition of nucleoside triphosphates (3 mM) to the assay medium, but nucleoside mono-and diphosphates were almost inactive. Pyrimidine nucleotides were less effective compared with purine nucleotides, among which GTP was the most effective. dATP and ATP analogues such as adenosine 5′-(β,γ-imino) triphosphate (AMP-PNP), adenosine 5′-(β,γ-methylene) triphosphate (AMP-PCP), etc. were inactive. ATP or GTP showed no additive effect on enzyme activity stimulated by dithiothreitol or bovine serum albumin.     

Biosynthesis of 5-(4''5''-dihydroxypentyl) uracil as a nucleoside triphosphate in bacteriophage SP15-infected Bacillus subtilis.

The nucleoside triphosphate of 5-(4',5'-dihydroxypentyl)uracil (DHPU) was detected in the acid-soluble extract from bacteriophage SP15-infected Bacillus subtilis W23. No uracil was found in the DNA of either replicating or mature phage. Labeled thymidine added during phage DNA synthesis was incorporated into phage DNA. The presence of DHPU as a nucleoside triphosphate in the acid-soluble pool and the incorporation of thymidine into phage DNA suggest that both DHPU and thymine are incorporated into SP15 DNA via their nucleoside triphosphates. 5-Fluorodeoxyuridine inhibited biosynthesis of SP15 DNA, and this inhibition was reversed by thymidine, resulting in the synthesis of a DNA containing reduced amounts of fully modified DHPU. It is proposed that 5-fluorodeoxyuridine, or its metabolic product, inhibits a step in the biosynthetic pathway to the nucleoside triphosphate of DHPU.     

Pestivirus NS3 (p80) protein possesses RNA helicase activity.

The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed.     

末端脱氧核苷酸转移酶在生物传感及核酸合成领域的应用*

末端脱氧核苷酸转移酶(terminal deoxynucleotidyl transferase, TdT)是聚合酶X家族中的一员,与典型的DNA聚合酶不同,TdT以恒温的无模板依赖的方式催化脱氧核糖核苷三磷酸(dNTP)聚合到寡核苷酸的3'羟基端来合成DNA。并且TdT对底物的耐受性高具有聚合修饰型dNTP的能力,如荧光修饰的dNTP、生物素修饰的dNTP,甚至人工碱基均可作为其良好底物。TdT的这些生化特性使其被广泛的应用在生物传感和核酸合成领域中,促进了许多基于核酸的工具和方法的发展,并为酶促从头合成DNA技术的发展奠定基础。介绍了TdT的性质,重点总结了它在其介导的生物检测技术、核酸的修饰技术以及酶促合成DNA技术三个方面的核心作用、目前面临的挑战以及未来研究的方向,以期促进TdT在生物传感器和核酸合成中的进一步应用。     

Induction of SOS functions: Regulation of proteolytic activity of E. coil RecA protein by interaction with DNA and nucleoside triphosphate

Damage to cellular DNA or interruption of chromosomal DNA synthesis leads to induction of the SOS functions in E. coli. The immediate agent of induction is the RecA protein, which proteolytically cleaves and inactivates repressors, leading to induction of genes they control. RecA protein modified by tif mutations allows expression of SOS functions in the absence of inducing treatments. We show here that tif-mutant RecA protein is more efficient than wild-type RecA protein in interacting with DNA and nucleoside triphosphate. This result suggests that formation of a complex with DNA and nucleoside triphosphate is the critical event that activates RecA protein to destroy repressors after SOS-inducing treatments, and that damage to cellular DNA promotes this reaction by providing single-stranded DNA or an active nucleoside triphosphate or both. Since dATP is the most effective nucleoside triphosphate in promoting repressor cleavage, we suggest that it is the natural cofactor of RecA protein in vivo.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.     

Synthesis of amide-linked [(3')CH2CO-NH(5')] nucleoside analogues of small oligonucleotides

We report syntheses of new amide-linked (di-penta)nucleoside analogues of antisense oligonucleotide components. Solution-phase coupling of 3'-(carboxymethyl)-3'-deoxy- and 5'-amino-5'-deoxynucleoside derivatives provides amide dimers. Activated [3'-(carboxymethyl)-3'-deoxy] units with a 5'-azido-5'-deoxy function provide "masked" 5'-amino-5'-deoxy residues for chain extension, and a 5'-O-DMT-protected unit provides the 5'-terminus for attachment to a phosphodiester linkage.     

Binding of Cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii. Identification of dimethylbenzimidazole as the axial ligand

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.     

Comparative effects of the 5'-triphosphates of 9-beta-(2'-azido-2'-deoxy-D-arabinofuranosyl)adenine and 9-beta-D-arabinofuranosyladenine on DNA polymerases from L1210 leukemia cells.

9-beta-(2'-Azido-2'-deoxy-D-arabiofuranosyl)adenine (arazide) causes greater and significantly more persistent inhibition of [3H]-thymidine incorporation into the DNA of neoplastic cells than the related agent 9-beta-D-arabinofuranosyladenine (araA). To elucidate the mechanism(s) responsible, we compared the effects of arazide and araA 5'-triphosphates on DNA polymerases alpha and beta of L1210 leukemia cells. Both nucleoside triphosphate analogs inhibited DNA polymerase alpha activity by competing with dATP; only araATP was inhibitory to DNA polymerase beta. Arazide triphosphate was at least four times more active than araATP as an inhibitor of DNA polymerase alpha. Preincubation of DNA polymerase alpha with either agent did not result in enzyme inactivation. The results suggest that interference with DNA polymerase alpha activity by arazide triphosphate may be in part responsible for the inhibition of DNA synthesis produced by arazide in neoplastic cells.