Effects of a high-affinity antibody fragment on DNA polymerase reactions near a (6-4) photoproduct site

Pyrimidine (6-4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6-4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41-mer DNA containing a (6-4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases alpha, beta and delta (pol alpha, pol beta and pol delta). None of these DNA polymerases were able to bypass the (6-4) photoproduct and elongation terminated at or near the 3'-pyrimidone of the photoproduct, depending on the dNTP concentration. When a single-chain Fv (scFv) with high affinity for the (6-4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3'-pyrimidone by Klenow fragment, pol alpha, pol beta or pol delta, respectively. These results suggest that the scFv can bind to the template DNA containing a (6-4) photoproduct and inhibit extension reactions by polymerases.     

Thermodynamic Impact of Abasic Sites on Simulated Translesion DNA Synthesis

Loss of a base in DNA and the creation of an abasic (apurinic/apyrimidinic, AP) site is a frequent lesion that may occur spontaneously, or as a consequence of the action of DNA‐damaging agents. The AP lesion is mutagenic or lethal if not repaired. We report a systematic thermodynamic investigation by differential scanning calorimetry on the evolution, during primer extension, of a model AP site in chemically simulated DNA translesion synthesis. Incorporation of dAMP (deoxyadenosine monophosphate), as well as dTMP (deoxythymidine monophosphate), opposite an AP site is enthalpically unfavorable, although incorporation of dTMP is more enthalpically unfavorable than that of dAMP. This finding is in a good agreement with experimental data showing that AP sites block various DNA polymerases of eukaryotic and prokaryotic origin and that, if bypassed, dAMP is preferentially inserted, whereas insertion of dTMP is less likely. The results emphasize the importance of thermodynamic contributions to the insertion of nucleotides opposite an AP site by DNA polymerases.     

Biochemical Characterization of Translesion Synthesis by Sulfolobus acidocaldarius DNA Polymerases

To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarchaeota, two DNA polymerases of B family(polB1 and polB3), and one DNA polymerase of Y family(polIV) were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polB1(Saci_1537) and polB3(Saci_0074) possessed DNA polymerase and 3' to 5' exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV(Saci_0554) was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn²⁺ and Mg²⁺. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KCl with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.     

Hydrogen bonding contributes to the selectivity of nucleotide incorporation opposite an oxidized abasic lesion

The ability of DNA polymerases to maintain the integrity of the genome even after it has been structurally altered is vital. There is considerable interest in determining the structural properties of the DNA template that polymerases recognize when determining which nucleotide to add to a nascent strand. Mechanistic, synthetic, and structural chemistries have been used to study how DNA polymerase activity is affected by size, shape, pi-stacking, and hydrogen bonds of the template molecules. Herein, we probe the structural aspects of abasic lesions that result in their distinct coding potential in Escherichia coli despite lacking a Watson-Crick base. In particular, we investigate why bypass of 2-deoxyribonolactone (L) results in significant amounts of dG incorporation opposite the lesion, whereas other abasic lesions (e.g., AP) adhere to the "A-rule". Experiments using synthetic analogues reveal that DNA polymerase V bypasses L and increased levels of dG incorporation result from a hydrogen bonding interaction between the carbonyl oxygen and dG. These results show that a DNA polymerase utilizes hydrogen bonding as one structural parameter when decoding an abasic lesion.     

Guanine-thymine intrastrand cross-linked lesion containing oligonucleotides: from chemical synthesis to in vitro enzymatic replication

An intrastrand cross-link lesion, in which two neighboring nucleobases are covalently tethered, has been site-specifically synthesized into defined sequence oligonucleotides in order to perform in vitro replication studies using either bacterial replicative or translesional synthesis polymerases. The investigated tandem base lesion that involves a cross-link between the methylene group of thymine and the C8 of an adjacent guanine residue has been prepared by UV-photolysis under anaerobic condition of the photolabile precursor 5-(phenylthiomethyl)-2'-deoxyuridine that has been site-specifically incorporated into a 9-mer oligonucleotide. After ligation, the lesion-containing modified oligonucleotide was used as a DNA template in primer extension reactions catalyzed by several DNA polymerases including the fragment Klenow exo-(Kf-) of E. coli polymerase I, the Thermus aquaticus polymerase (Taq pol) and the E. coli translesional DNA polymerase Pol IV (dinB). It was found that the primer extension reaction was stopped after the incorporation of the correct nucleotide dAMP opposite the 3'-thymine residue of guanine(C8-CH2) thymine lesion by Kf- and Pol IV; however it was noted that the efficiency of the nucleotide incorporation was reduced. In contrast, the Taq polymerase was totally blocked at the nucleotide preceding the tandem lesion. These results are strongly suggestive that the present intrastrand cross-link lesion, if not repaired, would constitute a blocking lesion for prokaryotic DNA polymerases, being likely lethal for the cell.     

Unnatural substrate repertoire of A, B, and X family DNA polymerases

As part of an effort to develop unnatural base pairs that are stable and replicable in DNA, we examined the ability of five different polymerases to replicate DNA containing four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs. The unnatural pairs were developed based on intensive studies using the Klenow fragment of DNA polymerase I from E. coli (Kf) and found to be recognized to varying degrees. The five additional polymerases characterized here include family A polymerases from bacteriophage T7 and Thermus aquaticus, family B polymerases from Thermococcus litoralis and Thermococcus 9(o)N-7, and the family X polymerase, human polymerase beta. While we find that some aspects of unnatural base pair recognition are conserved among the polymerases, for example, the pair formed between two d3FB nucleotides is typically well recognized, the detailed recognition of most of the unnatural base pairs is generally polymerase dependent. In contrast, we find that the pair formed between d5SICS and dMMO2 is generally well recognized by all of the polymerases examined, suggesting that the determinants of efficient and general recognition are contained within the geometric and electronic structure of these unnatural nucleobases themselves. The data suggest that while the d3FB:d3FB pair is sufficiently well recognized by several of the polymerases for in vitro applications, the d5SICS:dMMO2 heteropair is likely uniquely promising for in vivo use. T7-mediated replication is especially noteworthy due to strong mispair discrimination.     

Solution structure and conformational dynamics of deoxyxylonucleic acids (dXNA): an orthogonal nucleic acid candidate

Orthogonal nucleic acids are chemically modified nucleic acid polymers that are unable to transfer information with natural nucleic acids and thus can be used in synthetic biology to store and transfer genetic information independently. Recently, it was proposed that xylose-DNA (dXNA) can be considered to be a potential candidate for an orthogonal system. Herein, we present the structure in solution and conformational analysis of two self-complementary, fully modified dXNA oligonucleotides, as determined by CD and NMR spectroscopy. These studies are the initial experimental proof of the structural orthogonality of dXNAs. In aqueous solution, dXNA duplexes predominantly form a linear ladderlike (type-1) structure. This is the first example of a furanose nucleic acid that adopts a ladderlike structure. In the presence of salt, an equilibrium exists between two types of duplex form. The corresponding nucleoside triphosphates (dXNTPs) were synthesized and evaluated for their ability to be incorporated into a growing DNA chain by using several natural and mutant DNA polymerases. Despite the structural orthogonality of dXNA, DNA polymerase β mutant is able to incorporate the dXNTPs, showing DNA-dependent dXNA polymerase activity.     

Base pairing and replicative processing of the formamidopyrimidine-dG DNA lesion

The 2,6-diamino-4-hydroxy-5-formamidopyrimidine of 2'-deoxyguanosine (FaPydG) is one of the major DNA lesions found after oxidative stress in cells. To clarify the base pairing and coding potential of this major DNA lesion with the aim to estimate its mutagenic effect, we prepared oligonucleotides containing a cyclopentane based analogue of the DNA lesion (cFaPydG). In addition, oligonucleotides containing the cyclopentane analogue of 2'-deoxyguanosine (cdG), and oligonucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were synthesized. The thermodynamic stability of duplexes containing these building blocks and all canonical counterbases were determined by concentration dependent melting-point measurements (van't Hoff plots). The data reveal that cFaPydG greatly destabilizes a DNA duplex (DeltaDeltaG degrees (298K) approximately 2-4 kcal mol(-1)). The optimal base pairing partner for the cFaPydG lesion is dC. Investigation of duplexes containing dG and cdG shows that the effect of substituting the deoxyribose by a cyclopentane moiety is marginal. The data also provide strong evidence that the FaPydG lesion is unable to form a base pair with dA. Our computational studies indicate that the syn-conformation required for base pairing with dA is energetically unfavorable. This is in contrast to 8-oxodG for which the syn-conformation represents the energetic minimum. Kinetic primer extension studies using S. cerevisiae Pol eta reveal that cFaPydG is replicated in an error-free fashion. dC is inserted 2-3 orders of magnitude more efficiently than dT or dA, showing that FaPydG is a lesion which retains the coding potential of dG. This is also in contrast to 8-oxodG, for which base pairing with dC and dA was established.     

The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases

Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of molecular biological and biotechnological approaches. Different candidates have been developed whereby alternative hydrogen-bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base-pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as experimental studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications.     

Opposed steric constraints in human DNA polymerase beta and E. coli DNA polymerase I

DNA polymerase selectivity is crucial for the survival of any living species, yet varies significantly among different DNA polymerases. Errors within DNA polymerase-catalyzed DNA synthesis result from the insertion of noncanonical nucleotides and extension of misaligned DNA substrates. The substrate binding characteristics among DNA polymerases are believed to vary in properties such as shape and tightness of the binding pocket, which might account for the observed differences in fidelity. Here, we employed 4'-alkylated nucleotides and primer strands bearing 4'-alkylated nucleotides at the 3'-terminal position as steric probes to investigate differential active site properties of human DNA polymerase beta (Pol beta) and the 3'-->5'-exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (KF(exo-)). Transient kinetic measurements indicate that both enzymes vary significantly in active site tightness at both positions. While small 4'-methyl and -ethyl modifications of the nucleoside triphosphate perturb Pol beta catalysis, extension of modified primer strands is only marginally affected. Just the opposite was observed for KF(exo-). Here, incorporation of the modified nucleotides is only slightly reduced, whereas size augmentation of the 3'-terminal nucleotide in the primer reduces the catalytic efficiency by more than 7000- and 260,000-fold, respectively. NMR studies support the notion that the observed effects derive from enzyme substrate interactions rather than inherent properties of the modified substrates. These findings are consistent with the observed differential capability of the investigated DNA polymerases in fidelity such as processing misaligned DNA substrates. The results presented provide direct evidence for the involvement of varied steric effects among different DNA polymerases on their fidelity.     

Electrostatic energy analysis of 8-oxoguanine DNA lesion—molecular dynamics study

One nanosecond molecular dynamics (MD) simulation was performed for two DNA segments each composed of 30 base pairs. In one DNA segment the native guanines at nucleotides positions 17 and 19 were replaced with two 8-oxoguanines (8-oxoG) (8-oxoG is mutagenic DNA oxo-lesion). The analysis of results was focused on the electrostatic energy that is supposed to be significant factor causing the disruption of DNA base stacking in DNA duplex and may also serve as a signal toward the repair enzyme informing the presence of the lesion. The repulsive interaction between 8-oxoG and the entire DNA molecule was observed, which caused the extrahelical position of 8-oxoG (position 19). The repulsive electrostatic interaction between both 8-oxoG lesions contributed to the flipping out of one 8-oxoG and to the local instability of the lesioned DNA region. The electrostatic potential at the surface of DNA close to the lesions has more negative value than the same region on the native DNA. This electrostatic potential may signal presence of the lesion to the repair enzyme. In the simulation of native DNA segment, no significant structural changes were observed and B-DNA structure was well preserved throughout the MD simulation.     

Polymerase evolution: efforts toward expansion of the genetic code

Genetic information is encoded by, but potentially not limited to, a four-letter alphabet. A variety of predominantly hydrophobic nucleobase analogues that form self-pairs in DNA have been examined as third base pair candidates. For example, the PICS self-pair is both stable in duplex DNA and synthesized by some wild-type polymerases with reasonable efficiency. These efforts to expand the genetic code are expected to be facilitated by optimizing both the unnatural nucleobase analogues and the polymerases that replicate them. Here, we report the use of an activity-based selection system to evolve a DNA polymerase that more efficiently replicates DNA containing the PICS self-pair. The selection system is based on the co-display on phage of DNA polymerase libraries and a DNA substrate containing the self-pair. Only polymerases that accept the unnatural substrate incorporate a biotin-dUTP to the attached primer and may then be isolated on a streptavidin solid support. A mutant of Sf polymerase, P2, was evolved which both inserts dPICSTP opposite dPICS in the template and extends the unnatural primer terminus by incorporation of the next correct natural dNTP, where the parental enzyme catalyzes neither step at detectable rates. P2 was found to be a triple mutant of Sf, with the mutations F598I, I614F, and Q489H. The evolved properties of P2, as well as the observed mutations, are consistent with an increased affinity for the DNA primer-template containing the self-pair.     

Direct Sensing of 5‐Methylcytosine by Polymerase Chain Reaction

The epigenetic control of genes by the methylation of cytosine resulting in 5‐methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′‐mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation‐specific PCR approach directly from untreated human genomic DNA.     

Unnatural Cytosine Bases Recognized as Thymines by DNA Polymerases by the Formation of the Watson–Crick Geometry

The emergence of unnatural DNA bases provides opportunities to demystify the mechanisms by which DNA polymerases faithfully decode chemical information on the template. It was previously shown that two unnatural cytosine bases (termed “M‐fC” and “I‐fC”), which are chemical labeling adducts of the epigenetic base 5‐formylcytosine, can induce C‐to‐T transition during DNA amplification. However, how DNA polymerases recognize such unnatural cytosine bases remains enigmatic. Herein, crystal structures of unnatural cytosine bases pairing to dA/dG in the KlenTaq polymerase‐host–guest complex system and pairing to dATP in the KlenTaq polymerase active site were determined. Both M‐fC and I‐fC base pair with dA/dATP, but not with dG, in a Watson–Crick geometry. This study reveals that the formation of the Watson–Crick geometry, which may be enabled by the A‐rule, is important for the recognition of unnatural cytosines.     

Effective form of sulfoquinovosyldiacyglycerol (SQDG) vesicles for DNA polymerase inhibition

Sulfoquinovosyldiacyglycerol (SQDG) has a wide range of biological activities that make it an attractive compound for the development of new drugs. Chemically synthesized beta-SQDG-C(18:0) (1,2-di-O-stearoyl-3-O-(6-deoxy-6-sulfo-beta-d-glucopyranosyl)-sn-glycerol), for example, has a potent inhibitory effect on DNA polymerases. We investigated the properties of the vesicle form of beta-SQDG-C(18:0) as the monomer has low solubility in water. The structure of the beta-SQDG-C(18:0) vesicles are highly influenced by NaCl concentration in preparation process. At low NaCl concentrations, the beta-SQDG-C(18:0) vesicles have high surface curvature and form small unilamellar vesicles. Increases in NaCl concentration, resulted in decreased surface curvature and a tendency for beta-SQDG-C(18:0) to form large multilamellar vesicles. The small unilamellar vesicles showed a potent inhibitory effect on DNA polymerase beta, whereas the large multilamellar vesicles had no such effect. We investigated further the relationship between vesicle size and activity by preparing smaller vesicles (262, 99 and 43 nm in diameter) using an extrusion technique. These smaller vesicles had a greater inhibitory effect on DNA polymerase beta activity than non-extruded vesicles. beta-SQDG-C(18:0) vesicles, especially those of small size, were effective in DNA polymerase inhibition and are expected to have high applicability in DNA polymerase study.     

Tuning the reaction site for enzyme-free primer-extension reactions through small molecule substituents

The replication of genetic information relies on the template-directed extension of DNA primers catalyzed by polymerases. The active sites of polymerases accept four different substrates and ensure fidelity and processivity for each of them. Because of the pivotal role of catalyzed primer extension for life, it is important to better understand this reaction on a molecular level. Here we present results from primer-extension reactions performed with chemical systems that show high reactivity in the absence of polymerases. Small molecular caps linked to the 5'-terminus of templates are shown to enhance the rate and selectivity of primer extension driven by 2-methylimidazolides as activated monomers for any of the four different templating bases (A, C, G, and T). The most consistent effect is provided by a stilbene carboxamide residue, rather than larger aromatic or aliphatic substituents. Up to 20-fold rate enhancements were achieved for the reactions at the terminus of the template. The preference for a medium size cap can be explained by competing interactions with both the oligonucleotides and the incoming deoxynucleotide. The data also show that there is no particularly intractable problem in combining promiscuity with fidelity. Exploratory experiments involving a longer template and a downstream-binding strand with a 5'-cap show up to 38-fold rate acceleration over the same reaction templated by a single overhanging nucleotide.     

Ag(I) ion mediated formation of a C-A mispair by DNA polymerases

Silver turns up the A-C: In the presence of Ag(I) ions, a DNA polymerase incorporated deoxyadenosine (from dATP) at the site opposite cytosine in the template strand to afford the full-length product (see scheme), meaning that DNA polymerases prefer a C-Ag(I)-A base pair to the more thermodynamically stable C-Ag(I)-C base pair.     

Quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization

The nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis. We examine here the possible reaction pathways of a Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4), for the correct insertion of dCTP opposite 8-oxoguanine using the quantum mechanics/molecular mechanics (QM/MM) approach, both from a chemistry-competent state and a crystal closed state. The latter examination is important for understanding pre-chemistry barriers to interpret the entire enzyme mechanism, since the crystal closed state is not an ideal state for initiating the chemical reaction. The most favorable reaction path involves initial deprotonation of O3'H via two bridging water molecules to O1A, overcoming an overall potential energy barrier of approximately 20.0 kcal/mol. The proton on O1A-P(alpha) then migrates to the gamma-phosphate oxygen of the incoming nucleotide as O3' attacks P(alpha), and the P(alpha)-O3A bond breaks. The other possible pathway in which the O3'H proton is transferred directly to O1A on P(alpha) has an overall energy barrier of 25.0 kcal/mol. In both reaction paths, the rate-limiting step is the initial deprotonation, and the trigonal-bipyramidal configuration for P(alpha) occurs during the concerted bond formation (O3'-P(alpha)) and breaking (P(alpha)-O3A), indicating the associative nature of the chemical reaction. In contrast, the Dpo4/DNA complex with an imperfect active-site geometry corresponding to the crystal state must overcome a much higher activation energy barrier (29.0 kcal/mol) to achieve a tightly organized site due to hindered O3'H deprotonation stemming from larger distances and distorted conformation of the proton acceptors. This significant difference demonstrates that the pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the primer terminus environment. Compared to the higher fidelity DNA polymerase beta from the X-family, Dpo4 has a higher chemical reaction barrier (20.0 vs 15.0 kcal/mol) due to the more solvent-exposed active site.