Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7‐Substituted 7‐Deazaguanine Nucleobases

Previous studies of polymerase synthesis of base‐modified DNAs and their cleavage by restriction enzymes have mostly related only to 5‐substituted pyrimidine and 7‐substituted 7‐deazaadenine nucleotides. Here we report the synthesis of a series of 7‐substituted 7‐deazaguanine 2′‐deoxyribonucleoside 5′‐O‐triphosphates (dG^RTPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dG^RTPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7‐substituted 7‐deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage.     

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.     

1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA

In order to establish the Diels–Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4‐triazine‐modified 2′‐deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4‐triazine group was attached at the 5‐position of 2′‐deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2′‐deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN‐modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4‐triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5‐tetrazine for postsynthetic labeling by the Diels–Alder reaction with inverse electron demand.     

Synthesis of C8‐N‐Arylamine‐Modified 2′‐Deoxyguanosine‐5′‐Triphosphates and Their Effects on Primer Extension by DNA Polymerases

C8‐N‐arylamine adducts of 2′‐deoxyguanosine (2′‐dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8‐N‐acetyl‐N‐arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8‐N‐acetyl‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates and C8‐NH‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8‐dG adducts were studied in primer‐extension assays by using Klenow fragment exo^? of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N‐acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates in chemical carcinogenesis.     

Enzymatic Polymerization of Phosphonate Nucleosides

5′‐O‐Phosphonomethyl‐2′‐deoxyadenosine (PMdA) proved to be a good substrate of the Therminator polymerase. In this article, we investigated whether the A, C, T and U analogues of this phosphonate nucleoside (PMdN) series can function as substrates of natural DNA polymerases. PMdT and PMdU could only be polymerized enzymatically to a limited extent. Nevertheless, PMdA and PMdC could be incorporated into a DNA duplex with complete chain elongation by all the DNA polymerases tested. A mixed sequence of four nucleotides containing modified C, T and A residues could be obtained with the Vent(exo^?) and Therminator polymerases. The kinetic values for the incorporation of PMdA by Vent(exo^?) polymerase were determined; a reduced K_M value was found for the incorporation of PMdA compared to the natural substrate. Future polymerase directed evolution studies will allow us to select an enzyme with a heightened capacity to process these modified DNA building blocks into modified strands.     

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.     

6‐Substituted 2‐Aminopurine‐2′‐deoxyribonucleoside 5′‐Triphosphates that Trace Cytosine Methylation

Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2′‐deoxy 5‐methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O⁶‐alkylated 2′‐deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6‐substituted 2‐aminopurine‐2′‐deoxyribonucleoside 5′‐triphosphates modified at position 6 with various functionalities. We found that sensing of 5‐methylation by this class of nucleotides is more general, not being restricted to O⁶‐alkyl modification of dGTP but also applying to other functionalities.     

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.     

In Vivo Expression of Genetic Information from Phosphoramidate–DNA

Chemically modified genes and genomes with customized properties will become a valuable tool in numerous fields, including synthetic biology, biotechnology, and medicine. These genetic materials are meant to store and exchange information with DNA and RNA while tuning their functionality. Herein, we outline the development of an alternative genetic system carrying phosphoramidate linkages that successfully propagates genetic information in bacteria and at the same time is labile to acidic conditions. The P3′→N5′ phosphoramidate-containing DNA (PN-DNA) was enzymatically synthesized by using 5′-amino-2′,5′-deoxycytidine 5′-N-triphosphates (NH-dCTPs) as substrates for DNA polymerases and employed to encode antibiotic resistance in Escherichia coli. The resulting PN-DNA can be efficiently destroyed by mild acidic conditions, whereas an unmodified counterpart remains intact. A cloning strategy was proposed for assembling modified fragments into a genome. This method can be of interest to scientists working in the field of orthogonal nucleic acid genes and genomes.     

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.     

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.     

Generation of a mono-ubiquitinated PCNA mimic by click chemistry

Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high‐fidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono‐ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site‐specifically mono‐ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site‐specifically by the Cu^I‐catalyzed azide–alkyne cycloaddition. Finally, we show that the synthetic PCNA–Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA–Ub than to PCNA.     

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.     

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.     

Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5’-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.     

Protein Interactions in the T7 DNA Replisome Facilitate DNA Damage Bypass

The DNA replisome inevitably encounters DNA damage during DNA replication. The T7 DNA replisome contains a DNA polymerase (gp5), the processivity factor thioredoxin (trx), a helicase‐primase (gp4), and a ssDNA‐binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated strand‐displacement DNA synthesis past 8‐oxoG or O⁶‐MeG lesions at the synthetic DNA fork by the T7 DNA replisome. DNA damage does not obviously affect the binding affinities between helicase, polymerase, and DNA fork. Relative to unmodified G, both 8‐oxoG and O⁶‐MeG—as well as GC‐rich template sequence clusters—inhibit strand‐displacement DNA synthesis and produce partial extension products. Relative to the gp4 ΔC‐tail, gp4 promotes DNA damage bypass. The presence of gp2.5 also promotes it. Thus, the interactions of polymerase with helicase and ssDNA‐binding protein facilitate DNA damage bypass. Accessory proteins in other complicated DNA replisomes also facilitate bypassing DNA damage in similar manner. This work provides new mechanistic information relating to DNA damage bypass by the DNA replisome.