Incorporation of 2'-deoxy-2'-isonucleoside 5'-triphosphates (iNTPs) into DNA by A- and B-family DNA polymerases with different recognition mechanisms

Recently, α-L-threofuranosyl nucleoside 3'-triphosphates (tNTPs) have been reported to be incorporated into DNA by DNA polymerases. Isonucleosides especially the 2'-deoxy-2'-isonucleosides, would be considered regioisomers of α-L-threofuranosyl nucleosides. Therefore, we investigated the synthesis of 2'-deoxy-2'-isonucleoside 5'-triphosphates (iNTPs) having the four natural nucleobases and their incorporation into primer-template duplexes consisting of oligonucleotides containing natural 2'-deoxyribonucleosides and 2'-deoxy-2'-isonucleosides by using primer-extension reactions. We found that Klenow fragment (exo-; an A-family DNA polymerase) has strict recognition of the shape of nucleoside 5'-triphosphates and Therminator (a B-family DNA polymerase) has strict recognition of the shape of primer-template complexes, especially two base pairs upstream of the primer 3' terminus.     

Varied active-site constraints in the klenow fragment of E. coli DNA polymerase I and the lesion-bypass Dbh DNA polymerase

We report on comparative pre-steady-state kinetic analyses of exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment, KF-) and the archaeal Y-family DinB homologue (Dbh) of Sulfolobus solfataricus. We used size-augmented sugar-modified thymidine-5'-triphosphate (T(R)TP) analogues to test the effects of steric constraints in the active sites of the polymerases. These nucleotides serve as models for study of DNA polymerases exhibiting both relatively high and low intrinsic selectivity. Substitution of a hydrogen atom at the 4'-position in the nucleotide analogue by a methyl group reduces the maximum rate of nucleotide incorporation by about 40-fold for KF- and about twelve fold for Dbh. Increasing the size to an ethyl group leads to a further twofold reduction in the rates of incorporation for both enzymes. Interestingly, the affinity of KF- for the modified nucleotides is only marginally affected, which would indicate no discrimination during the binding step. Dbh even has a higher affinity for the modified analogues than it does for the natural substrate. Misincorporation of either TTP or T(Me)TP opposite a G template causes a drastic decline in incorporation rates for both enzymes. At the same time, the binding affinities of KF- for these nucleotides drop by about 16- and fourfold, respectively, whereas Dbh shows only a twofold reduction. Available structural data for ternary complexes of relevant DNA polymerases indicate that both enzymes make close contacts with the sugar moiety of the dNTP. Thus, the varied proficiencies of the two enzymes in processing the size-augmented probes indicate varied flexibility of the enzymes' active sites and support the notion of active site tightness being a criterion for 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).     

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.     

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.     

Mechanism of Error‐Free Bypass of the Environmental Carcinogen N‐(2′‐Deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone Adduct by Human DNA Polymerase η

The environmental pollutant 3‐nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8‐dG‐ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error‐free manner by the human Y‐family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol?DNA?dCTP complex between a C8‐dG‐ABA‐containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error‐free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra‐nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson–Crick pair with the incoming dCTP.     

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.     

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.      

An attempt to unify the structure of polymerases

With the great availability of sequences from RNA- and DNA-dependentRNA and DNA polymerases, it has become possible to delineatea few highly conserved regions for various polymerase types.In this work a DNA polymerase sequence from bacteriophage SPO2was found to be homologous to the polymerase domain of the Klenowfragment of polymerase I from Escherichia coli, which is knownto be closely related to those from Staphylococcus pneumoniae,Thermits aquaticus and bacteriophages T7 and T5. The alignmentof the SPO2 polymerase with the other five sequences considerablynarrowed the conserved motifs in these proteins. Three of themotifs matched reasonably all the conserved motifs of anotherDNA polymerase type, characterized by human polymerase a. Itis also possible to find these three motifs in monomeric DNA-dependentRNA polymerases and two of them in DNA polymerase ßand DNA terminal transferases. These latter two motifs alsomatched two of the four motifs recently identified in 84 RNA-dependentpolymerases. From the known tertiary architecture of the Klenowfragment of E.coli pol I, a spatial arrangement can be impliedfor these motifs. In addition, numerous biochemical experimentssuggesting a role for the motifs in a common function (dNTPbinding) also support these inferences. This speculative hypothesis,attempting to unify polymerase structure at least locally, ifnot globally, under the pol I fold, should provide a usefulmodel to direct mutagenesis experiments to probe template andsubstrate specificity in polymerases.     

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.     

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.     

The Multiple Cellular Roles of SMUG1 in Genome Maintenance and Cancer

Single-strand selective monofunctional uracil DNA glycosylase 1 (SMUG1) works to remove uracil and certain oxidized bases from DNA during base excision repair (BER). This review provides a historical characterization of SMUG1 and 5-hydroxymethyl-2′-deoxyuridine (5-hmdU) one important substrate of this enzyme. Biochemical and structural analyses provide remarkable insight into the mechanism of this glycosylase: SMUG1 has a unique helical wedge that influences damage recognition during repair. Rodent studies suggest that, while SMUG1 shares substrate specificity with another uracil glycosylase UNG2, loss of SMUG1 can have unique cellular phenotypes. This review highlights the multiple roles SMUG1 may play in preserving genome stability, and how the loss of SMUG1 activity may promote cancer. Finally, we discuss recent studies indicating SMUG1 has moonlighting functions beyond BER, playing a critical role in RNA processing including the RNA component of telomerase.     

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.     

Characterization of a Novel Mesophilic CTP-Dependent Riboflavin Kinase and Rational Engineering to Create Its Thermostable Homologues**

Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.     

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.     

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.     

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.     

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.     

Synthesis of C8-arylamine-modified 2'-deoxyadenosine phosphoramidites and their site-specific incorporation into oligonucleotides

Adducts of C8-(N-acetyl)-arylamines and 2'-deoxyadenosine were synthesised by palladium-catalysed C--N cross-coupling chemistry. These 2'-dA adducts were converted into the corresponding 3'-phosphoramidites and site-specifically incorporated into DNA oligonucleotides, which were characterised by mass spectrometry, UV thermal-stability assays and circular dichroism. These modified oligonucleotides were also used in EcoRI restriction assays and in primer-extension studies with three different DNA polymerases. The incorporation of the 2'-dA lesion close to the EcoRI restriction site dramatically reduced the susceptibility of the DNA strand to cleavage; this indicates a significant local distortion of the DNA double helix. The incorporation of the acetylated C8-2'-dA-phosphoramidites into 20-mer oligonucleotides failed, however, because the N-acetyl group was lost during the deprotection process. Instead the corresponding C8-NH-2'-dA-modified oligonucleotides were obtained. The effect of the C8-NH-arylamine-dA lesion on the replication by DNA polymerases was clearly dependent both on the polymerase used and on the arylamine-dA damage.