The use of non-natural nucleotides to probe template-independent DNA synthesis

The vast majority of DNA polymerases use the complementary templating strand of DNA to guide each nucleotide incorporation. There are instances, however, in which polymerases can efficiently incorporate nucleotides in the absence of templating information. This process, known as translesion DNA synthesis, can alter the proper genetic code of an organism. To further elucidate the mechanism of template-independent DNA synthesis, we monitored the incorporation of various nucleotides at the "blunt-end" of duplex DNA by the high-fidelity bacteriophage T4 DNA polymerase. Although natural nucleotides are not incorporated at the blunt-end, a limited subset of non-natural indolyl analogues containing extensive pi-electron surface areas are efficiently utilized by the T4 DNA polymerase. These analogues possess high binding affinities that are remarkably similar to those measured during incorporation opposite an abasic site. In contrast, the k(pol) values are significantly lower during blunt-end extension when compared to incorporation opposite an abasic site. These kinetic differences suggest that the single-stranded region of the DNA template plays an important role during polymerization through stacking interactions with downstream bases, interactions with key amino acid residues, or both. In addition, we demonstrate that terminal deoxynucleotide transferase, a template-independent enzyme, can efficiently incorporate many of these non-natural nucleotides. However, that this unique polymerase cannot extend large, bulky non-natural nucleotides suggests that elongation is limited by steric constraints imposed by structural features present within the polymerase. Regardless, the kinetic data obtained from using either DNA polymerase indicate that template-independent synthesis can occur without the contributions of hydrogen-bonding interactions and suggest that pi-electron interactions play an important role in polymerization efficiency when templating information is not present.     

Directed DNA polymerase evolution: effects of mutations in motif C on the mismatch-extension selectivity of thermus aquaticus DNA polymerase

The selectivity of DNA polymerases for processing the canonical nucleotide and DNA substrate in favor of the noncanonical ones is the key to the integrity of the genome of every living species and to many biotechnological applications. The inborn ability of most DNA polymerases to abort efficient extension of mismatched DNA substrates adds to the overall DNA polymerase selectivity. DNA polymerases have been grouped into families according to their sequence. Within family A DNA polymerases, six motifs that come into contact with the substrates and form the active site have been discovered to be evolutionary highly conserved. Here we present results obtained from amino acid randomization within one motif, motif C, of thermostable Thermus aquaticus DNA polymerase. We have identified several distinct mutation patterns that increase the selectivity of mismatch extension. These results might lead to direct applications such as allele-specific PCR, as demonstrated by real-time PCR experiments and add to our understanding of DNA polymerase selectivity.     

Enzymatic Incorporation of Modified Purine Nucleotides in DNA

A series of nucleotide analogues, with a hypoxanthine base moiety (8‐aminohypoxanthine, 1‐methyl‐8‐aminohypoxanthine, and 8‐oxohypoxanthine), together with 5‐methylisocytosine were tested as potential pairing partners of N⁸‐glycosylated nucleotides with an 8‐azaguanine or 8‐aza‐9‐deazaguanine base moiety by using DNA polymerases (incorporation studies). The best results were obtained with the 5‐methylisocytosine nucleotide followed by the 1‐methyl‐8‐aminohypoxanthine nucleotide. The experiments demonstrated that small differences in the structure (8‐azaguanine versus 8‐aza‐9‐deazaguanine) might lead to significant differences in recognition efficiency and selectivity, base pairing by Hoogsteen recognition at the polymerase level is possible, 8‐aza‐9‐deazaguanine represents a self‐complementary base pair, and a correlation exists between in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions were observed).     

A parallel synthesis scheme for generating libraries of DNA polymerase substrates and inhibitors

We report a combinatorial approach aimed at producing in a single step a large family of nucleoside triphosphate derivatives that could be tested for their ability to be substrates for DNA polymerases. We propose as a unique triphosphate building block a nucleotide with a hydrazine function anchored to an imidazole ring. Condensation between the 5'-triphosphate derivative of 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH(2))TP) and any aldehyde or ketone, followed by reduction of the intermediate hydrazones dXmTP, resulted in the corresponding hydrazides (dXnTP). Following this scheme, a series of aldehydes having various aromatic parts yielded a number of adducts dY(NHR)TP. Vent (exo-) DNA polymerase is found to be able to catalyse the single incorporation of these bulky triphosphate derivatives. Subsequent extensions of the modified pairs with canonical triphosphates resulted mainly in abortive elongations at primer+2, except after the incorporation of dY(NHben)TP and, to a lesser extent, dY(NHphe)TP opposite C. These results illustrate the potential of this parallel synthetic scheme for generating new substrates or inhibitors of replication in a single step.     

Fluorescent analysis of translesion DNA synthesis by using a novel, non-natural nucleotide analogue

The replication of damaged DNA is a promutagenic process that can lead to disease development. This report evaluates the dynamics of nucleotide incorporation opposite an abasic site, a commonly formed DNA lesion, by using two fluorescent nucleotide analogues, 2-aminopurine deoxyribose triphosphate (2-APTP) and 5-phenylindole deoxyribose triphosphate (5-PhITP). In both cases, the kinetics of incorporation were compared by using a 32P-radiolabel extension assay versus a fluorescence-quenching assay. Although 2-APTP is efficiently incorporated opposite a templating nucleobase (thymine), the kinetics for incorporation opposite an abasic site are significantly slower. The lower catalytic efficiency hinders its use as a probe to study translesion DNA synthesis. In contrast, the rate constant for the incorporation of 5-PhITP opposite the DNA lesion is 100-fold faster than that for 2-APTP. Nearly identical kinetic parameters are obtained from fluorescence quenching or the 32P-radiolabel assay. Surprisingly, distinct differences in the kinetics of 5-PhITP incorporation opposite the DNA lesion are detected when using either bacteriophage T4 DNA polymerase or the Escherichia coli Klenow fragment. These differences suggest that the dynamics of nucleotide incorporation opposite an abasic site are polymerase-dependent. Collectively, these data indicate that 5-PhITP can be used to perform real-time analyses of translesion DNA synthesis as well as to functionally probe differences in polymerase function.     

Bacteriophage-Encoded DNA Polymerases—Beyond the Traditional View of Polymerase Activities

DNA polymerases are enzymes capable of synthesizing DNA. They are involved in replication of genomes of all cellular organisms as well as in processes of DNA repair and genetic recombination. However, DNA polymerases can also be encoded by viruses, including bacteriophages, and such enzymes are involved in viral DNA replication. DNA synthesizing enzymes are grouped in several families according to their structures and functions. Nevertheless, there are examples of bacteriophage-encoded DNA polymerases which are significantly different from other known enzymes capable of catalyzing synthesis of DNA. These differences are both structural and functional, indicating a huge biodiversity of bacteriophages and specific properties of their enzymes which had to evolve under certain conditions, selecting unusual properties of the enzymes which are nonetheless crucial for survival of these viruses, propagating as special kinds of obligatory parasites. In this review, we present a brief overview on DNA polymerases, and then we discuss unusual properties of different bacteriophage-encoded enzymes, such as those able to initiate DNA synthesis using the protein-priming mechanisms or even start this process without any primer, as well as able to incorporate untypical nucleotides. Apart from being extremely interesting examples of biochemical biodiversity, bacteriophage-encoded DNA polymerases can also be useful tools in genetic engineering and biotechnology.     

Learning from directed evolution: Thermus aquaticus DNA polymerase mutants with translesion synthesis activity

DNA is being constantly damaged by endo- and exogenous agents such as reactive oxygen species, chemicals, radioactivity, and ultraviolet radiation. Additionally, DNA is inherently labile, and this can result in, for example, the spontaneous hydrolysis of the glycosidic bond that connects the sugar and the nucleobase moieties in DNA; this results in abasic sites. It has long been obscure how cells achieve DNA synthesis past these lesions, and only recently has it been discovered that several specialized DNA polymerases are involved in translesion synthesis. The underlying mechanisms that render one DNA polymerase competent in translesion synthesis while another DNA polymerase fails are still indistinct. Recently two variants of Taq DNA polymerase that exhibited higher lesion bypass ability than the wild-type enzyme were identified by directed-evolution approaches. Strikingly, in both approaches it was independently found that substitution of a single nonpolar amino acid side chain by a cationic side chain increases the capability of translesion synthesis. Here, we combined both mutations in a single enzyme. We found that the KlenTaq DNA polymerase that bore both mutations superseded the wild-type as well as the respective single mutants in translesion-bypass proficiency. Further insights in the molecular basis of the detected gain of translesion-synthesis function were obtained by structural studies of DNA polymerase variants caught in processing canonical and damaged substrates. We found that increased positive charge of the surface potential in the area proximal to the negatively charged substrates promotes translesion synthesis by KlenTaq DNA polymerase, an enzyme that has very limited naturally evolved capability to perform translesion synthesis. Since expanded positively charged surface potential areas are also found in naturally evolved translesion DNA polymerases, our results underscore the impact of charge on the proficiency of naturally evolved translesion DNA polymerases.     

Polymerase Amplification,Cloning, and Gene Expression of Benzo‐Homologous “yDNA” Base Pairs

A widened DNA base‐pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the “yDNA” system, pairs are homologated by addition of a benzene ring, which yields (in the present study) benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15–20 % of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, and yielded functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture.     

Protein Domain Specific Covalent Inhibition of Human DNA Polymerase β

DNA polymerase β (Pol β) is a frequently overexpressed and/or mutated bifunctional repair enzyme. Pol β possesses polymerase and lyase active sites, that are employed in two steps of base excision repair. Pol β is an attractive therapeutic target for which there is a need for inhibitors. Two mechanistically inspired covalent inhibitors ( 1 , IC₅₀=21.0 μM; 9 , IC₅₀=18.7 μM) that modify lysine residues in different Pol β active sites are characterized. Despite modifying lysine residues in different active sites, 1 and 9 inactivate the polymerase and lyase activities of Pol β. Fluorescence anisotropy experiments indicate that they do so by preventing DNA binding. Inhibitors 1 and 9 provide the basis for a general approach to preparing domain selective inhibitors of bifunctional polymerases. Such molecules could prove to be useful tools for studying the role of wild type and mutant forms of Pol β and other polymerases in DNA repair.     

2′‐Deoxyribonucleoside Phosphoramidate Triphosphate Analogues as Alternative Substrates for E. coli Polymerase III

Thermostable bacterial polymerases like Taq, Therminator and Vent exo^? are able to perform DNA synthesis by using modified DNA precursors, a property that is exploited in several therapeutic and biotechnological applications. Viral polymerases are also known to accept modified substrates, and this has proven crucial in the development of antiviral therapies. However, non‐thermostable polymerases of bacterial origin, or engineered variants, that have similar substrate tolerance and could be used for synthetic biology purposes remain to be identified. We have identified the α subunit of Escherichia coli polymerase III (Pol III α) as a bacterial polymerase that is able to recognise and process as substrates several pyrophosphate‐modified dATP analogues in place of its natural substrate dATP for template‐directed DNA synthesis. A number of dATP analogues featuring a modified pyrophosphate group were able to serve as substrates during enzymatic DNA synthesis by Pol III α. Features such as the presence of potentially chelating chemical groups and the size and spatial flexibility of the chemical structure seem to be of major importance for the modified leaving group to play its role during the enzymatic reaction. In addition, we could establish that if the pyrophosphate group is altered, deoxynucleotide incorporation proceeds with an efficiency varying with the nature of the nucleobase. Our results represent a great step towards the achievement of a system of artificial DNA synthesis hosted by E. coli and involving the use of altered nucleotide precursors for nucleic acid synthesis.     

Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases

One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.     

Keeping uracil out of DNA: physiological role, structure and catalytic mechanism of dUTPases

The thymine-uracil exchange constitutes one of the major chemical differences between DNA and RNA. Although these two bases form the same Watson-Crick base pairs with adenine and are equivalent for both information storage and transmission, uracil incorporation in DNA is usually a mistake that needs to be excised. There are two ways for uracil to appear in DNA: thymine replacement and cytosine deamination. Most DNA polymerases readily incorporate dUMP as well as dTMP depending solely on the availability of the d(U/T)TP building block nucleotides. Cytosine deamination results in mutagenic U:G mismatches that must be excised. The repair system, however, also excises U from U:A "normal" pairs. It is therefore crucial to limit thymine-replacing uracils.dUTP is constantly produced in the pyrimidine biosynthesis network. To prevent uracil incorporation into DNA, representatives of the dUTP nucleotidohydrolase (dUTPase) enzyme family eliminate excess dUTP. This Account describes recent studies that have provided important detailed insights into the structure and function of these essential enzymes.dUTPases typically possess exquisite specificity and display an intriguing homotrimer active site architecture. Conserved residues from all three monomers contribute to each of the three active sites within the dUTPase. Although even dUTPases from evolutionarily distant species possess similar structural and functional traits, in a few cases, a monomer dUTPase mimics the trimer structure through an unusual folding pattern. Catalysis proceeds by way of an SN2 mechanism; a water molecule initiates in-line nucleophilic attack. The dUTPase binding pocket is highly specific for uracil. Phosphate chain coordination involves Mg2+ and is analogous to that of DNA polymerases. Because of conformational changes in the enzyme during catalysis, most crystal structures have not resolved the residues in the C-terminus. However, recent high-resolution structures are beginning to provide in-depth structural information about this region of the protein.The dUTPase family of enzymes also shows promise as novel targets for anticancer and antimicrobial therapies. dUTPase is upregulated in human tumor cells. In addition, dUTPase inhibitors could also fight infectious diseases such as malaria and tuberculosis. In these respective pathogens, Plasmodium falciparum and Mycobacterium tuberculosis, the biosynthesis of dTMP relies exclusively on dUTPase activity.     

Structural studies on an inhibitory antibody against Thermus aquaticus DNA polymerase suggest mode of inhibition

TP7, an antibody against Thermus aquaticus DNA polymerase I (TaqP), is used as a thermolabile switch in 'hot start' variations of PCR to minimize non-specific amplification events. Earlier studies have established that TP7 binds to the polymerase domain of TaqP, competes with primer template complex for binding and is a potent inhibitor of the polymerase activity of TaqP. We report crystallographic determination of the structure of an Fab fragment of TP7 and computational docking of the structure with the known three-dimensional structure of the enzyme. Our observations strongly suggest that the origin of inhibitory ability of TP7 is its binding to enzyme residues involved in DNA binding and polymerization mechanism. Although criteria unbiased by extant biochemical data have been used in identification of a putative solution, the resulting complex offers an eminently plausible structural explanation of biochemical observations. The results presented are of general significance for interpretation of docking experiments and in design of small molecular inhibitors of TaqP, that are not structurally similar to substrates, for use in PCR. Structural and functional similarities noted among DNA polymerases, and the fact that several DNA polymerases are pharmacological targets, make discovery of non-substrate based inhibitors of additional importance.      

Modified DNA bearing 5(methoxycarbonylmethyl)-2'-deoxyuridine: preparation by PCR with thermophilic DNA polymerase and postsynthetic derivatization

A thymidine analogue bearing a methyl ester at the C5 position was accepted as a substrate by the thermophilic family B DNA polymerases, KOD Dash, Pwo, and Vent(exo-), to form the corresponding PCR product, but not by the thermophilic family A DNA polymerases, Taq, Tth, and T7 thermosequenase. Modified DNA containing this analogue was prepared by PCR on a large scale with KOD Dash DNA polymerase and 5(methoxycarbonylmethyl)-2'-deoxyuridine 5'-triphosphate as a substrate. The methyl ester of the modified DNA was further allowed to react with tris(2-aminoethyl)amine or histamine by an ester-amide exchange reaction to form the corresponding derivatized DNA bearing a tris(2-aminoethyl)amine or histamine moiety. Hydrolysis of the methyl ester of the modified DNA gave a functionalized DNA bearing an anionic carboxyl group. The derivatized DNA could act as a template for the PCR with KOD Dash DNA polymerase and the natural 2'-deoxythymidine 5'-triphosphate or the modified thymidine analogue as a substrate. The postsynthetic derivatization of the modified DNA may expand the variety of structurally modified DNA produced by PCR.     

Transient N4-Acyl-DNA Protection against Cleavage by Restriction Endonucleases

A set of five N⁴-acyl-modified 2′-deoxycytidine 5′-triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N⁴-acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N⁴-acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high-fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N⁴ modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one-pot procedure that eliminated additional purification stages. It is suggested that N⁴-modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.     

Guanine-rich DNA nanocircles for the synthesis and characterization of long cytosine-rich telomeric DNAs

Short synthetic oligonucleotides derived from the human telomeric repeat have been studied recently for their ability to fold into four-stranded structures that are thought to be important to their biological function. Because telomeric DNAs are several kilobases in length, however, their folding might well be affected by cooperative or high-order interactions in these long sequences. Here, we present a new molecular system that allows for easy synthesis of very long stretches of the cytosine-rich strand of human telomeric DNA. Small circular DNAs composed of the G-rich sequence of human telomeres were prepared and used as templates in a rolling-circle replication mechanism. To facilitate the synthesis of the repetitive G-rich circles, an orthogonal base-protection strategy that made use of dimethylformamidine-protected guanine nucleobases was developed. Nanometer-scale circles ranging in size from 42 to 54 nucleotides were prepared. Subsequently, we tested the action of various DNA polymerases on these circular templates, and identified DNA Pol I (Klenow fragment) and T7 DNA polymerase as enzymes that are able to generate very long, C-rich telomeric DNA strands. Purification and initial structural examination of these C-rich polymeric products revealed evidence of a folded structure in the polymer.     

Incorporation of Nucleoside Probes Opposite O6‐Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4: Importance of Hydrogen Bonding

O⁶‐Methylguanine (O⁶‐MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low‐fidelity Y‐family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y‐family polymerases bypass DNA adducts. Previous work showed that Dpo4‐mediated dTTP incorporation is favored opposite O⁶‐MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶‐MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶‐MeG in a DNA template. To this end, we utilized a new fluorescence‐based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶‐MeG by Dpo4. In single‐dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶‐MeG. Moreover, pyrimidine‐based dNTPs were generally better incorporated than purine‐based syn‐conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2‐position of dCTP increase the selectivity for incorporation opposite O⁶‐MeG without a significant loss of efficiency.