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

Abstract

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.

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

“…More specifically, Thr664, which is situated closest to the thiophenyl moiety of dDs and to both dNaM and d5SICSTP , shifts upwards by 0.6 Å (measured at Cα atoms) in KlenTaq dDs–dPxTP and 0.9 Å in KlenTaq dNaM–d5SICSTP . To accommodate the propynyldiol moiety of dPxTP , Arg660, located at the N‐terminus of the O‐helix, shifts away in a similar way as already observed for other KlenTaq structures with modified substrates (Figure E) . Owing to the shift, an interaction of Arg660 with the 3′‐primer end is lost.…”

“…More specifically,T hr664, which is situatedc losest to the thiophenyl moiety of dDs andt ob oth dNaM and d5SICSTP,s hifts upwards by 0.6 (measured at Ca atoms) in KlenTaq dDs-dPxTP and 0.9 in KlenTaq dNaM-d5SICSTP .T oa ccommodate the propynyldiol moiety of dPxTP,A rg660, located at the N-terminus of the O-helix,s hifts away in as imilarw ay as already observed for other KlenTaq structures with modified substrates ( Figure 7E). [72][73][74] Owing to the shift, an interaction of Figure 8B). Apparently,t he finger domain cannot close as tightly as in the natural complex, which could explain why the studied hydrophobic artificial base pairs are still formed with somewhat diminished efficiency compared with the natural counterparts.…”

Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates

“…The authors also identified alkene-modified nucleotides with more efficient incorporation than their native counterparts as supported by crystal structures of catalytically competent complexes. Why the polymerase preferentially incorporates alkene-modified nucleotides over alkyne-modified nucleotides remains unclear, however [19]. Using inspiration from the accommodating polymerase active site, a more comprehensive study with BF observed aromatic bases of dNTPs with strong affinity for an active-site arginine, which interacts with the dNTP via a π-cation interaction [24].…”

“…Experiments with such substrates have generated insights into the enzyme’s preferences and formed the basis for sequencing technologies (e.g., Sanger sequencing using ddNTPs) [8,9,11–13]. Base-modified dNTP analogs can probe molecular recognition of both the incoming dNTP substrate and an appropriately substituted template base [5,14–19]. This review will survey the key biophysical techniques that have employed dNTP base and ribose analogs and the insights gained from these studies.…”

Cover Picture: 1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA (ChemBioChem 15/2017)

“…For the subsequentp rimere xtension experiments with the 1,2,4-triazine-modified 2'-deoxyuridinet riphosphate 8,V ent-(exo-), Deep Vent(exo-), and Homo Klen Ta q( New England Bio-Labs), and KOD XL DNA (Merck) polymerases were screened because they typically accept modificationsi nt he 5-position [ Communications DOI:1 0.1002/cbic.201700185 of pyrimidines. [15] The primer extension experiments were first performed using the primer P1 (Scheme 2) that contained a fluoresceinl abel at the 5'-terminus in order to follow the elongation product by fluorescencei maging of gels after electrophoresis by using the green channelo ft he detector.T emplate T1 was chosen for the initial screening with standing-start experiments. T2-T4 were designed for multiple incorporationso f 9 in running-start experiments.…”