Enzymatic incorporation in DNA of 1,5-anhydrohexitol nucleotides

The ability of several DNA polymerases to catalyze the template-directed synthesis of duplex oligonucleotides containing a base pair between a nucleotide with anhydrohexitol ring and its natural complement has been investigated. All DNA polymerases were able to accept the chemically synthesized anhydrohexitol triphosphate as substrate and to catalyze the incorporation of one anhydrohexitol nucleotide. However, only family B DNA polymerases succeeded in elongating the primer after the incorporation of an anhydrohexitol nucleotide. In this family, Vent (exo(-)) DNA polymerase is the most successful one and was therefore selected for further investigation. Results revealed that at high enzyme concentrations six hATPs could be incorporated; however, a selective incorporation proved only feasible under experimental conditions where no more than two analogues could be inserted. Also the synthesis of a mixed HNA-DNA sequence was examined. Kinetic parameters for incorporation of one anhydrohexitol adenine nucleoside were similar to those of its natural analogue.     

Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases

Although DNA polymerase eta (Pol eta) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains. Here, we examine the role in Saccharomyces cerevisiae Pol eta of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation. We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably. The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family. Furthermore, we note that tyrosine 64 and arginine 67 are functionally equivalent to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, respectively.     

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases

Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OH-dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase η, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.     

Kinetics and mechanism of DNA repair. Evaluation of caged compounds for use in studies of u.v.-induced DNA repair.

Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the gamma-phosphate are placed in situ by permeabilizing cells by exposure to hypo-osmotic medium. The nucleoside triphosphate is released from the cage by a 351 nm u.v. laser pulse whence it may incorporate in the growing chain of DNA induced by the excision-repair process and terminate chain elongation. If the photoreleased dideoxynucleoside triphosphate is isotopically labelled in the alpha-phosphate position the break is trapped and labelled. Incorporation of radioactivity into trichloroacetic acid insoluble material in these experiments confirms their potential for use in studies of the kinetics of mammalian cell DNA repair.     

Recognition of HNA and 1,5-anhydrohexitol nucleotides by DNA metabolizing enzymes

Hexitol nucleic acids (HNA) as well as their 1,5-anhydrohexitol triphosphate building blocks were evaluated for their ability to be recognized by several DNA metabolizing enzymes. It was found that RNA polymerases can recognize the triphosphate of the adenine analogue. However, only the incorporation of a maximum of three consecutive building block analogues was possible under the applied experimental conditions. Terminal transferase was more successful succeeding in the elongation of a DNA primer with a maximum of 15 1,5-anhydrohexitol purine nucleotides. Furthermore, it was observed that the 1,5-anhydroaltritol triphosphate analogue of adenosine was a poor substrate for terminal transferase and that HNA could not act as a primer for this enzyme. Likewise, HNA did not function as a template for restriction enzymes, ligases or methylases.     

Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala⁵¹⁰ and Asn⁵¹³, play differential roles in dNTP selectivity. Specifically, Ala⁵¹⁰ and Asn⁵¹³ facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.     

Herpes simplex virus type 1 and human DNA polymerase interactions with 2'-deoxyguanosine 5'-triphosphate analogues. Kinetics of incorporation into DNA and induction of inhibition

The ability of herpes simplex virus type 1 (HSV-1) DNA polymerase, HeLa polymerase alpha, and HeLa polymerase beta to utilize several dGTP analogues has been investigated using a defined synthetic template primer. The relative efficiencies of the triphosphates of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir triphosphate, ACVTP), 9-[(1,3-dihydroxy-2-propoxy)methyl] guanine (ganciclovir triphosphate, DHPGTP), and 2',3'-dideoxyguanosine (ddGTP) as substrates for the three polymerases were: HSV-1 polymerase, dGTP greater than ACVTP approximately equal to DHPGTP greater than ddGTP; polymerase alpha, dGTP greater than ACVTP approximately equal to DHPGTP much greater than ddGTP; polymerase beta, ddGTP greater than dGTP much greater than ACVTP approximately equal to DHPGTP. The potent inhibition of HSV-1 polymerase by ACVTP has been shown previously to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3' end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism was shown here to be a general mechanism for inhibition of polymerases by the obligate chain terminators, ACVTP and ddGTP. The ACVTP-induced inhibition was 30-fold more potent with HSV-1 polymerase than with polymerase alpha. This difference may contribute to the antiviral selectivity of this nucleotide analogue. The effect of ganciclovir monophosphate incorporation (a nonobligate chain terminator) on subsequent primer extension was also evaluated. With HSV-1 polymerase and polymerase alpha, although there was a considerable reduction in the efficiency of utilization of the 3'-DHPGMP-terminal primer, contrasting kinetic behavior was observed. With HSV-1 polymerase, insertion of DHPGTP resulted in a significant reduction in Vmax for subsequent nucleotide incorporations. In contrast, with polymerase alpha, a relatively small decrease in Vmax was accompanied by increased Km values for subsequent nucleotide incorporations.     

Increased stability of nucleic acids containing 7-deaza-guanosine and 7-deaza-adenosine may enable rapid DNA sequencing by matrix-assisted laser desorption mass spectrometry.

The use of matrix-assisted laser desorption mass spectrometry (MALDI-MS) has been suggested as an ultrafast readout of Sanger DNA sequencing ladders in a manner analogous to that used with sequencing gels. Currently, a serious limitation of MALDI-MS for the analysis of DNA results from the tendency for oligonucleotides to undergo facile fragmentation in the gas phase. The present study was undertaken to gain an understanding of the influence of various chemical structural features of purine bases on the stability of oligodeoxynucleotide ions produced by MALDI. The study focused on the stability of model compounds of the type d(TTTTTTTTTTXTTTTTTTTT TTTT), where T designates deoxythymidine and X a purine-containing 2'-deoxynucleotide. A variety of different purine derivatives were chosen as the base in the nucleotide X. The mass spectra of the model compounds containing 7-deaza analogues of guanine and adenine reveal a significantly increased stability compared to the 7-aza analogues under the conditions of MALDI-MS. The previously reported incorporation of the 7-deaza-2'-deoxy-adenosine triphosphate and the 7-deaza-2'-deoxy-guanosine triphosphate into DNA by polymerases suggests their use in a Sanger dideoxy sequencing experiment. The dideoxy termination products with the 7-deaza-purines instead of the 7-aza-purines might be sufficiently stable to allow separation and detection of the sequencing ladder by MALDI-MS. Thus, an ultrafast (seconds) read-out of DNA sequence may become feasible.     

Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase.

The interactions of azidothymidine triphosphate, the metabolically active form of the anti-AIDS drug azidothymidine (zidovudine), with the cellular DNA polymerases alpha, delta, and epsilon, as well as with the RNA primer-forming enzyme DNA primase were studied in vitro. DNA polymerase alpha was shown to incorporate azidothymidine monophosphate into a growing polynucleotide chain. This occurred 2000-fold slower than the incorporation of natural dTTP. Despite the ability of polymerase alpha to use azidothymidine triphosphate as an alternate substrate, this compound was only marginally inhibitory to the enzyme (Ki greater than 1 mM). Furthermore, the DNA primase activity associated with DNA polymerase alpha was barely inhibited by azidothymidine triphosphate (Ki greater than 1 mM). Inhibition was more pronounced for DNA polymerases delta and epsilon. The type of inhibition was competitive with respect to dTTP, with Ki values of 250 and 320 microM, respectively. No incorporation of azidothymidine monophosphate was detectable with these two DNA polymerases because their associated 3'- to 5'-exonuclease activities degraded primer molecules prior to any measurable elongation. Template-primer systems with a preformed 3'-azidothymidine-containing primer terminus inhibited the three replicative polymerases rather potently. DNA polymerase alpha was inhibited with a Ki of 150 nM and polymerases delta and epsilon with Ki values of 25 and 20 nM, respectively. The type of inhibition was competitive with respect to the unmodified substrate poly(dA).oligo(dT) for all DNA polymerases tested. Performed 3'-azidothymidine-containing primers hybridized to poly(dA) were rather resistant to degradation by the 3'- to 5'-exonuclease of DNA polymerases epsilon and more susceptible to the analogous activity that copurified with DNA polymerase delta. It is proposed that the repair of 3'-azidothymidine-containing primers might become rate-limiting for the process of DNA replication in cells that have been treated with azidothymidine triphosphate.     

Mechanistic investigation of the bypass of a bulky aromatic DNA adduct catalyzed by a Y-family DNA polymerase

3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG^C8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dG^C8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dG^C8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dG^C8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dG^C8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dG^C8-N-ABA bypass catalyzed by Dpo4.     

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol lambda; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol lambda wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol beta in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.     

Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase

Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent(A488L) DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.     

Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

We synthesized C5-modified analogs of 2′-deoxyuridine triphosphate and 2′-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates × 5 DNA polymerases × 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3′ end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.     

Template recognition and chain elongation in DNA synthesis in vitro.

DNA polymerase from Escherichia coli (Pol I) and from avian myeloblastosis virus (AMV polymerase) were compared for the manner in which they catalyze the polymerization of deoxynucleotides upon a variety of synthetic and natural templates. It was found that the rates of nucleotide incorporation with different natural RNAs were similar. Both polymerases have an associated RNA endonuclease which hydrolyses RNA templates containing double-stranded regions. This activity depends on the presence of the complementary deoxynucleoside triphosphates, and/or polymerization. Both enzymes copy natural DNA, which has been sonicated and treated with E. coli exonuclease III, at the same rate. However, avian myeloblastosis virus DNA polymerase, which has no associated DNA exonuclease activity, is unable to copy double-stranded DNA and copies DNAase-treated DNA only 10% as well as Pol I. Pol I copied all the homopolymers investigated at a greater rate than AMV polymerase with the exception of poly(C) · oligo(dG). However, the initial rate of chain elongation, as measured by gel electrophoresis, was the same for the two polymerases, approximately 300 nucleotides incorporated per minute. Template saturation experiments show a stoichiometric relationship between template and enzyme at optimal rates of nucleotide incorporation which suggests that all enzyme molecules are potential catalysts. Enzyme saturation experiments indicate that not all enzyme molecules are “effectively” bound to a template. Fewer AMV polymerase than Pol I molecules are functionally bound to a particular template. From these data, it is concluded that the two polymerases elongate DNA chains in a similar way and that the manner in which the polymerases bind to a particular template accounts for the discrepancies found in their turnover numbers.     

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase alpha

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo⁻) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment.     

Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacet-amidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc2-dUTP) have been produced to demonstrate the incorporation of redox labels into DNA by polymerases. Cyclic voltammetry indicates that the ferrocenyl moieties display reversible redox behaviour in aqueous buffer with E_1/2 values of 398 (Fc1-dUTP) and 260 mV (Fc2-dUTP) versus Ag/AgCl. Primer extension by the proofreading enzymes Klenow fragment and T4 DNA polymerase shows that Fc1-dUTP is efficiently incorporated into DNA during synthesis, including incorporation of two successive modified nucleotides. Production of a 998 bp amplicon by Tth DNA polymerase demonstrates that Fc1-dUTP is also a satisfactory substrate for PCR. Despite its structural similarity, Fc2-dUTP acts predominantly as a terminator with the polymerases employed here. UV melting analysis of a 37mer duplex containing five Fc1-dU residues reveals that the labelled nucleotide introduces only a modest helix destabilisation, with T_m = 71 versus 75°C for the corresponding natural construct. Modified DNA is detected at femtomole levels using a HPLC system with a coulometric detector. The availability of simple and effective enzymatic labelling strategies should promote the further development of electrochemical detection in nucleic acid analysis.     

Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli

The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.