Abstract

Obtaining semisynthetic microorganisms that exploit the information density of “hachimoji” DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the “hachimoji” P:Z nucleobase pair with a similar efficiency to that seen for Watson–Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the “hachimoji” Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.

Highlights

  • We report a series of molecular dynamics (MD) simulations on eight binary complexes that test the idea that amino acid substitutions in the KlenTaq variant give rise to increased flexibility and reveal how altered dynamical motions might contribute to the increased efficiency of P:Z nucleobase incorporation by the KlenTaq variant

  • We tested the hypothesis that the four amino acid substitutions in the KlenTaq variant exert their effects by modifying dynamical motions in the binary complex[21] using four MD simulations: wild type (WT) KlenTaq bound to Watson-Crick DNA and P:Z-containing DNA duplexes, and the KlenTaq variant bound to the same Watson-Crick and artificially expanded genetic information systems (AEGIS) DNA duplexes

  • Turning the effects of individual residue substitutions on the structural properties of the template/primer duplex about the non-natural P:Z nucleobase pair in the AEGIS DNA, we find that the CZ dinucleotide slide and twist distributions observed for the M444V, P527A, and E832V KlenTaq variants are essentially identical to those seen for the WT polymerase

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Summary

Introduction

Identifying DNA polymerases capable of catalyzing the incorporation of P:Z nucleobase pairs with efficiencies comparable to those that replicate Watson-Crick DNA is a necessary pre-requisite to realizing the promise of expanded genetic alphabets.[15,16] A variety of library generation and selection strategies have been developed to re-engineer the fidelity of DNA polymerases,[17,18,19] including the large (Klenow) fragment of Thermus aquaticus DNA polymerase I, which lacks the Nterminal 5’-3’ exonuclease domain.[20] a compartmentalized self-replication strategy[21,22] was used to obtain a KlenTaq variant capable of incorporating dZTP opposite a P nucleobase in the template strand with a greatly improved efficiency relative to the wild type (WT) precursor (Fig. 1).[23] The evolved KlenTaq variant contains four amino acid replacements (M444V, P527A, D551E and E832V; Fig. 1), all of which are distal to the active site. None of these residues interact directly with either the primer/template P:Z in the active site or with incoming nucleotide triphosphate (dZTP) in high resolution crystal structures of the pre- and post-incorporation complexes for this variant polymerase (PDB:5W6K and PDB:5W6Q, respectively).[24]

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