Structures of KlenTaq DNA Polymerase Caught While Incorporating C5-Modified Pyrimidine and C7-Modified 7-Deazapurine Nucleoside Triphosphates

Abstract

The capability of DNA polymerases to accept chemically modified nucleotides is of paramount importance for many biotechnological applications. Although these analogues are widely used, the structural basis for the acceptance of the unnatural nucleotide surrogates has been only sparsely explored. Here we present in total six crystal structures of modified 2'-deoxynucleoside-5'-O-triphosphates (dNTPs) carrying modifications at the C5 positions of pyrimidines or C7 positions of 7-deazapurines in complex with a DNA polymerase and a primer/template complex. The modified dNTPs are in positions poised for catalysis leading to incorporation. These structural data provide insight into the mechanism of incorporation and acceptance of modified dNTPs. Our results open the door for rational design of modified nucleotides, which should offer great opportunities for future applications.

Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA

“…Ternary structures of KlenTaq (a polA family polymerase) have been solved with modified bases in the active site [32], [33]. In these studies, one of the main residues in the active site that is affected by the incorporation of C5 modified dNTPs is Arg 660 in the B-motif, which is found to be displaced to avoid steric clashes with the bulky C5 substituents.…”

N2‐Substituted 2′‐Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ

“…[2] Minor groove modifications are used to elucidate critical protein-DNA interactions, [3] while major groove modifications have proved to be useful in exploring polymerase enzymology [4] and cellular reactivity. [5] Inhibitors of viral reverse transcriptases are in clinical use for HIV, hepatitis B, and hepatitus C therapy. [6] Human DNA polymerase inhibitors are also in use for cancer chemotherapy.…”

“…Attachment of the ethynyl moiety to the benzyl group enables the use of copper(I)-catalyzed azide-alkyne cycloaddition chemistry to attach a fluorescent group to the nucleoside and thus determine the amount and location of EBdG in the cell. [5] The labelling of the nuclei is shown in Figure 4a–c, in which mouse embryonic fibroblasts (MEF) were incubated with 10 μM EBdG for 24h. The cells were fixed and the ethynyl groups were reacted with azido-FAM, and the nuclei identified with DAPI-staining of the DNA.…”

7‐Aryl‐7‐deazaadenine 2′‐Deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases than dATP in Competitive Incorporations

“…Most modified dNTPs were worse substrates for polymerases than the natural ones, [8] with the exception of 7-[(hydroxydecanoyl)aminopentynyl]-7-deaza-dATP, which was a slightly better substrate for KlenTaq polymerase than dATP, but the authors did not comment on it further. [9] However, this PAGE-based method can only be applied for the analysis of single nucleotide incorporation with bulky modifications, where the differences in mobility are distinctive. Therefore, a new method needed to be developed for the analysis of competitive primer extension and multiple incorporations (including PCR).…”