Microenvironment-Sensitive Fluorescent Nucleotide Probes from Benzofuran, Benzothiophene, and Selenophene as Substrates for DNA Polymerases.

DNA polymerases can process a wide variety of structurally diverse nucleotide substrates, but the molecular basis by which the analogs are processed is not completely understood. Here, we demonstrate the utility of environment-sensitive heterocycle-modified fluorescent nucleotide substrates in probing the incorporation mechanism of DNA polymerases in real time and at the atomic level. The nucleotide analogs containing a selenophene, benzofuran, or benzothiophene moiety at the C5 position of 2'-deoxyuridine are incorporated into oligonucleotides (ONs) with varying efficiency, which depends on the size of the heterocycle modification and the DNA polymerase sequence family used. KlenTaq (A family DNA polymerase) is sensitive to the size of the modification as it incorporates only one heterobicycle-modified nucleotide into the growing polymer, whereas it efficiently incorporates the selenophene-modified nucleotide analog at multiple positions. Notably, in the single nucleotide incorporation assay, irrespective of the heterocycle size, it exclusively adds a single nucleotide at the 3'-end of a primer, which enabled devising a simple two-step site-specific ON labeling technique. KOD and Vent(exo-) DNA polymerases, belonging to the B family, tolerate all the three modified nucleotides and produce ONs with multiple labels. Importantly, the benzofuran-modified nucleotide (BFdUTP) serves as an excellent reporter by providing real-time fluorescence readouts to monitor enzyme activity and estimate the binding events in the catalytic cycle. Further, a direct comparison of the incorporation profiles, fluorescence data, and crystal structure of a ternary complex of KlenTaq DNA polymerase with BFdUTP poised for catalysis provides a detailed understanding of the mechanism of incorporation of heterocycle-modified nucleotides.

Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase.

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5'-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.

The Structure of an Archaeal B-Family DNA Polymerase in Complex with a Chemically Modified Nucleotide.

Archaeal B-family DNA polymerases (DNA pols) are the driving force of cutting-edge biotechnological applications like next-generation sequencing. The acceptance of chemically modified nucleotides by DNA pols is key to these technologies. Until now, no structural data have been available for these DNA pols in complex with modified substrates, which could build the basis for understanding interactions between the enzyme and the chemically modified nucleotide and for the further development of next-generation nucleotides. For the first time, we crystallized an exonuclease-deficient variant of the wild-type B-family KOD DNA pol with a modified nucleotide in a closed, ternary complex. We also crystalized the A-family DNA pol KlenTaq with the same nucleotide. The reported structural data reveal how the protein and the DNA modulate two distinct conformations of the appended moiety in the A- and B-family DNA pols and how these influence the processing of the modified nucleotide. Overall, this study provides first insight into the interplay between B-family DNA pols and relevant modified 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.

Regioselective Cleavage of Thioether Linkages in Microcystin Conjugates.

Microcystins are cyanobacterial toxins that can be found in fresh and coastal waters during algal blooms. Microcystin contamination of water can cause severe poisoning of animals and humans. Quantification of these toxins in biological samples is complicated because a major proportion of microcystins is covalently linked to proteins through thioether bonds formed through a Michael-type addition of cysteine residues of proteins to an N-methyldehydroalanine residue in the microcystins. We investigated chemical methods that can be used to cleave such thioether bonds by means of an elimination reaction that leaves the microcystin backbone intact for subsequent analysis. The known reagent O-mesitylenesulfonylhydroxylamine (MSH) led to regioselective thioether cleavage, but a large excess of reagent was needed, thus making purification challenging. An unexpected side reaction observed during the investigation of the base-induced elimination inspired us to develop a new thioether-cleavage methodology based on the addition of propargylamine as a nucleophile that can trap the elimination product. This methodology could be successfully applied to the quantitative cleavage of a microcystin-LF-glutathione conjugate. The alkyne moiety introduced by this procedure offers the possibility for further reactions with azides by using click chemistry, which might be useful for the derivatization or isolation of microcystins.