Snapshot of a DNA polymerase while incorporating two consecutive C5-modified nucleotides.

Functional nucleotides are important in many cutting-edge biomolecular techniques. Often several modified nucleotides have to be incorporated consecutively. This structural study of KlenTaq DNA polymerase, a truncated form of Thermus aquaticus DNA polymerase, gives first insights how multiple modifications are processed by a DNA polymerase and, therefore, contribute to the understanding of these enzymes in their interplay with artificial substrates.

Interactions of non-polar and "Click-able" nucleotides in the confines of a DNA polymerase active site.

Modified nucleotides play a paramount role in many cutting-edge biomolecular techniques. The present structural study highlights the plasticity and flexibility of the active site of a DNA polymerase while incorporating non-polar "Click-able" nucleotide analogs and emphasizes new insights into rational design guidelines for modified nucleotides.

Amino acid templating mechanisms in selection of nucleotides opposite abasic sites by a family a DNA polymerase.

Cleavage of the N-glycosidic bond that connects the nucleobase to the backbone in DNA leads to abasic sites, the most frequent lesion under physiological conditions. Several DNA polymerases preferentially incorporate an A opposite this lesion, a phenomenon termed "A-rule." Accordingly, KlenTaq, the large fragment of Thermus aquaticus DNA polymerase I, incorporates a nucleotide opposite an abasic site with efficiencies of A > G > T > C. Here we provide structural insights into constraints of the active site during nucleotide selection opposite an abasic site. It appears that these confines govern the nucleotide selection mainly by interaction of the incoming nucleotide with Tyr-671. Depending on the nucleobase, the nucleotides are differently positioned opposite Tyr-671 resulting in different alignments of the functional groups that are required for bond formation. The distances between the α-phosphate and the 3'-primer terminus increases in the order A < G < T, which follows the order of incorporation efficiency. Additionally, a binary KlenTaq structure bound to DNA containing an abasic site indicates that binding of the nucleotide triggers a remarkable rearrangement of enzyme and DNA template. The ability to resolve the stacking arrangement might be dependent on the intrinsic properties of the respective nucleotide contributing to nucleotide selection. Furthermore, we studied the incorporation of a non-natural nucleotide opposite an abasic site. The nucleotide was often used in studying stacking effects in DNA polymerization. Here, no interaction with Tyr-761 as found for the natural nucleotides is observed, indicating a different reaction path for this non-natural nucleotide.

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.

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase.

Numerous 2'-deoxynucleoside triphosphates (dNTPs) that are functionalized with spacious modifications such as dyes and affinity tags like biotin are substrates for DNA polymerases. They are widely employed in many cutting-edge technologies like advanced DNA sequencing approaches, microarrays, and single molecule techniques. Modifications attached to the nucleobase are accepted by many DNA polymerases, and thus, dNTPs bearing nucleobase modifications are predominantly employed. When pyrimidines are used the modifications are almost exclusively at the C5 position to avoid disturbing of Watson-Crick base pairing ability. However, the detailed molecular mechanism by which C5 modifications are processed by a DNA polymerase is poorly understood. Here, we present the first crystal structures of a DNA polymerase from Thermus aquaticus processing two C5 modified substrates that are accepted by the enzyme with different efficiencies. The structures were obtained as ternary complex of the enzyme bound to primer/template duplex with the respective modified dNTP in position poised for catalysis leading to incorporation. Thus, the study provides insights into the incorporation mechanism of the modified nucleotides elucidating how bulky modifications are accepted by the enzyme. The structures show a varied degree of perturbation of the enzyme substrate complexes depending on the nature of the modifications suggesting design principles for future developments of modified substrates for DNA polymerases.

Replication through an abasic DNA lesion: structural basis for adenine selectivity.

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A-rule. In this study, we present X-ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non-instructive lesions by DNA polymerases at least from this sequence family.

Enzymatic synthesis of multi spin-labeled DNA.

Electron paramagnetic resonance (EPR) spectroscopy was applied extensively in studies of nucleic acid structures and dynamics. Two modified 2'-deoxyuridine triphosphates were synthesized bearing a spin label linked to the base by a rigid linker to ensure a tight coupling of spin label dynamics. The incorporation of both spin-labeled nucleotides could be shown in primer extension reactions in presence of DNA polymerases from eukaryotic, prokaryotic, and archaic origin. In further experiments we were able to introduce multiple spin labels during primer extension reactions.