The control of the discrimination between dNTP and rNTP in DNA and RNA polymerase

Yoon, Hanwool; Warshel, Arieh

doi:10.1002/prot.25104

Cited by 8 publications

(8 citation statements)

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“…S1). The EVB parameters were taken from the earlier study of T7 RNA polymerase (which has the same reacting atoms) . The protonation states were determined by calculating the pK a s with our coarse grained (CG) model for the residues within 12 Å from the simulation center.…”

Section: Methodsmentioning

confidence: 99%

“…The EVB parameters were taken from the earlier study of T7 RNA polymerase (which has the same reacting atoms). 21 The protonation states were determined by calculating the pK a s with our coarse grained (CG) model 22 for the residues within 12 Å from the simulation center. The effect of ionizable residues beyond this range were treated macroscopically using a large chargecharge dielectric.…”

Section: Methodsmentioning

confidence: 99%

“…The landscape of the reaction of pol h as a function of the reaction path from the reactant to the product state and the distances between the 3 rd Mg21 and the O2A of the incoming nucleotide. [Color figure can be viewed at wileyonlinelibrary.com]…”

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confidence: 99%

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Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis

Yoon

Warshel

2017

Proteins

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Pol η belongs to the important Y family of DNA polymerases that can catalyze translesion synthesis across sites of damaged DNA. This activity involves the reduced fidelity of Pol η for 8-oxo-7,8-dhyedro-2′-deoxoguanosin(8-oxoG). The fundamental interest in Pol η has grown recently with the demonstration of the importance of a 3rd Mg2+ ion. The current work explores both the fidelity of Pol η and the role of the 3rd metal ion, by using empirical valence bond (EVB) simulations. The simulations reproduce the observed trend in fidelity and shed a new light on the role of the 3rd metal ion. It is found that this ion does not lead to a major catalytic effect, but most probably plays an important role in reducing the product release barrier. Furthermore, it is concluded, in contrast to some implications, that the effect of this metal does not violate transition state theory, and the evaluation of the catalytic effect must conserve the molecular composition upon moving from the reactant to the transition state.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis

Yoon

Warshel

2017

Proteins

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“…Ucisik and Hammes‐Schiffer also reported binding free energy calculations using the more computationally expensive thermodynamic integration and reproduced the specificity of DNA Pol η for dATP versus dGTP opposite template T 183 . On the other hand, to rationalize the sugar selectivity of RB69 DNA Pol and T7 RNA Pol, Yoon and Warshel evaluated the change in

∆ G_{bind}^{TS}

upon mutating CTP to dCTP 184 . The FEP calculations suggested that the discrimination against CTP by RB69 DNA Pol is due to steric interaction (van der Waals contribution) between the 2′‐OH group and an active site Tyr (Y416), while the discrimination against dCTP by T7 RNA Pol is due to electrostatic effects.…”

Section: Computational Studies Of Polymerase Catalytic Mechanism and ...mentioning

confidence: 99%

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

Geronimo

Vidossich

Donati

et al. 2021

WIREs Comput Mol Sci

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DNA and RNA polymerases (Pols) are central to life, health, and biotechnology because they allow the flow of genetic information in biological systems. Importantly, Pol function and (de)regulation are linked to human diseases, notably cancer (DNA Pols) and viral infections (RNA Pols) such as COVID‐19. In addition, Pols are used in various applications such as synthesis of artificial genetic polymers and DNA amplification in molecular biology, medicine, and forensic analysis. Because of all of this, the field of Pols is an intense research area, in which computational studies contribute to elucidating experimentally inaccessible atomistic details of Pol function. In detail, Pols catalyze the replication, transcription, and repair of nucleic acids through the addition, via a nucleotidyl transfer reaction, of a nucleotide to the 3′‐end of the growing nucleic acid strand. Here, we analyze how computational methods, including force‐field‐based molecular dynamics, quantum mechanics/molecular mechanics, and free energy simulations, have advanced our understanding of Pols. We examine the complex interaction of chemical and physical events during Pol catalysis, like metal‐aided enzymatic reactions for nucleotide addition and large conformational rearrangements for substrate selection and binding. We also discuss the role of computational approaches in understanding the origin of Pol fidelity—the ability of Pols to incorporate the correct nucleotide that forms a Watson–Crick base pair with the base of the template nucleic acid strand. Finally, we explore how computations can accelerate the discovery of Pol‐targeting drugs and engineering of artificial Pols for synthetic and biotechnological applications. This article is categorized under: Structure and Mechanism > Reaction Mechanisms and Catalysis Structure and Mechanism > Computational Biochemistry and Biophysics Software > Molecular Modeling

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“…Transcription is a continuous process of synthesis of an RNA copy from the DNA templates, catalyzed by RNA polymerase (RNAP) with high fidelity. 18 , 19 , 20 T7 RNAP is a favorable bacteriophage RNAP. Containing a highly specific promoter recognition site and a nascent RNA-binding domain in its catalytic domain, T7 RNAP works self-sufficiently and drives transcription strongly without transcription factors.…”

Section: Introductionmentioning

confidence: 99%

Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

Liu

Kan

et al. 2020

Molecular Therapy - Nucleic Acids

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Due to highly enzymatic d -stereoselectivity, l -nucleotides ( l -2′-deoxynucleoside 5′-triphosphates [l -dNTPs]) are not natural targets of polymerases. In this study, we synthesized series of l -thymidine ( l -T)-modified DNA strands and evaluated the processivity of nucleotide incorporation for transcription by T7 RNA polymerase (RNAP) with an l -T-containing template. When single l -T was introduced into the transcribed region, transcription proceeded to afford the full-length transcript with different efficiencies. However, introduction of l -T into the non-transcribed region did not exhibit a noticeable change in the transcription efficiency. Surprisingly, when two consecutive or internal l -Ts were introduced into the transcribed region, no transcripts were detected. Compared to natural template, significant lags in NTP incorporation into the template T+4/N and T+7/N (where the number corresponds to the site of l -T position, and + means downstream of the transcribed region) were detected by kinetic analysis. Furthermore, affinity of template T+4/N was almost the same with T/N, whereas affinity of T+7/N was apparently increased. Furthermore, no mismatch opposite to l -T in the template was detected in transcription reactions via gel fidelity analysis. These results demonstrate the effects of chiral l -T in DNA on the efficiency and fidelity of RNA transcription mediated by T7 RNAP, which provides important knowledge about how mirror-image thymidine perturbs the flow of genetic information during RNA transcription and development of diseases caused by gene mutation.

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The control of the discrimination between dNTP and rNTP in DNA and RNA polymerase

Cited by 8 publications

References 58 publications

Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis

Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

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