Karin Betz

KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

Many candidate unnatural DNA base pairs have been developed, but surprisingly, some of the best replicated adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replicating one of the more promising pairs, dNaM-d5SICS, and show that efficient replication results from the polymerase itself inducing the required natural-like structure.

Structural Insights Into DNA Replication without Hydrogen Bonds.

The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the prechemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchemistry complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.

Structures of KOD and 9°N DNA Polymerases Complexed with Primer Template Duplex

Replicate it: Structures of KOD and 9°N DNA polymerases, two enzymes that are widely used to replicate DNA with highly modified nucleotides, were solved at high resolution in complex with primer/template duplex. The data elucidate substrate interaction of the two enzymes and pave the way for further optimisation of the enzymes and substrates.

Structures of DNA polymerases caught processing size-augmented nucleotide probes.

The integrity of the genome relies primarily on the ability of DNA polymerases to efficiently catalyze selective DNA synthesis according to the Watson–Crick rule in a templatedirected manner during DNA replication, repair, and recombination. Remarkably, some DNA polymerases achieve selective information transfer to the offspring in line with the Watson–Crick rule with intrinsic error rates as low as one mistake per one million synthesized nucleotides. This is far below the value that would be expected based on the energetic differences between canonical (i.e. Watson–Crick) and noncanonical nucleobase pairing. Geometric factors are widely cited to govern DNA polymerase selectivity. Thus, high-fidelity DNA polymerases are believed to mostly select the canonical nucleotide based on close steric complementarity of the nascent base pair to the geometry of the active site of the enzyme. Synthetic nucleotide analogues with tailored new properties are widely used to investigate the mechanisms that govern DNA polymerase selectivity. In this context nucleotides with hydrophobic nucleobase isosteres were developed to study the contribution of hydrogen bonding to DNA polymerase selectivity. 3] To investigate the steric effects in interactions between DNA polymerase and its substrate, we have developed 4’-alkyl-modified nucleotides with increasing steric demand. The modifications were introduced in the sugar residues since structural and functional data of DNA polymerases show that the sugar residues of nucleotides are involved in substrate recognition. These interactions may provide the enzymes with additional paths for achieving selectivity besides editing nucleobase geometry. Alkyl groups were employed since their effects on the hydrogen-bonding patterns and conformations of the nucleotides would be minimized. By employing these size-augmented nucleotides in functional DNA polymerase studies we could evaluate varied steric effects on DNA polymerase selectivity. Herein we report the first crystal structures of size-augmented 4’methylated and 4’-ethylated thymidine triphosphates in complex with a DNA polymerase. Significant mechanistic insights into nucleotide incorporation during DNA polymerization were derived from the high-resolution crystal structures reported by Waksman and colleagues of KlenTaq, an N-terminally truncated form of DNA polymerase from Thermus aquaticus. KlenTaq is a member of the family A DNA polymerases that play a role in prokaryotic and eukaryotic DNA replication and repair. Since data on the action of KlenTaq on 4’-alkylated thymidine-5’-triphosphates (dTTPs) is lacking, we first studied nucleotide incorporation by employing transient kinetic analysis with quench–flow technology (Table 1). Our data

Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of Alkene- versus Alkyne-Modified Nucleotides.

Efficient incorporation of modified nucleotides by DNA polymerases is essential for many cutting-edge biomolecular technologies. The present study compares the acceptance of either alkene- or alkyne-modified nucleotides by KlenTaq DNA polymerase and provides structural insights into how 7-deaza-adenosine and deoxyuridine with attached alkene-modifications are incorporated into the growing DNA strand. Thereby, we identified modified nucleotides that prove to be superior substrates for KlenTaq DNA polymerase compared with their natural analogues. The knowledge can be used to guide future design of functionalized nucleotide building blocks.

Structural Basis for Expansion of the Genetic Alphabet with an Artificial Nucleobase Pair.

Hydrophobic artificial nucleobase pairs without the ability to pair through hydrogen bonds are promising candidates to expand the genetic alphabet. The most successful nucleobase surrogates show little similarity to each other and their natural counterparts. It is thus puzzling how these unnatural molecules are processed by DNA polymerases that have evolved to efficiently work with the natural building blocks. Here, we report structural insight into the insertion of one of the most promising hydrophobic unnatural base pairs, the dDs-dPx pair, into a DNA strand by a DNA polymerase. We solved a crystal structure of KlenTaq DNA polymerase with a modified template/primer duplex bound to the unnatural triphosphate. The ternary complex shows that the artificial pair adopts a planar structure just like a natural nucleobase pair, and identifies features that might hint at the mechanisms accounting for the lower incorporation efficiency observed when processing the unnatural substrates.

Crystal structures of ternary complexes of archaeal B-family DNA polymerases.

Archaeal B-family polymerases drive biotechnology by accepting a wide substrate range of chemically modified nucleotides. By now no structural data for archaeal B-family DNA polymerases in a closed, ternary complex are available, which would be the basis for developing next generation nucleotides. We present the ternary crystal structures of KOD and 9°N DNA polymerases complexed with DNA and the incoming dATP. The structures reveal a third metal ion in the active site, which was so far only observed for the eukaryotic B-family DNA polymerase δ and no other B-family DNA polymerase. The structures reveal a wide inner channel and numerous interactions with the template strand that provide space for modifications within the enzyme and may account for the high processivity, respectively. The crystal structures provide insights into the superiority over other DNA polymerases concerning the acceptance of modified nucleotides.