Cardiff University | Prifysgol Caerdydd ORCA
Online Research @ Cardiff 
WelshClear Cookie - decide language by browser settings

Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase

Ouaray, Zahra, Singh, Isha, Georgiadis, Millie M. and Richards, Nigel G. J. 2020. Building better enzymes: molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase. Protein Science 29 (2) 10.1002/pro.3762

[img]
Preview
PDF - Published Version
Available under License Creative Commons Attribution.

Download (10MB) | Preview

Abstract

Obtaining semi‐synthetic 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 a series of 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 non‐natural nucleobases thereby limiting the number that need to be characterized by experiment.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Advanced Research Computing @ Cardiff (ARCCA)
Publisher: Wiley
ISSN: 0961-8368
Funders: BBSRC
Date of First Compliant Deposit: 5 November 2019
Date of Acceptance: 23 October 2019
Last Modified: 24 Mar 2020 15:30
URI: http://orca.cf.ac.uk/id/eprint/126594

Actions (repository staff only)

Edit Item Edit Item

Downloads

Downloads per month over past year

View more statistics