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WIREs Comput Mol Sci
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Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

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Abstract 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
Crystal structures of (a) DNA polymerase (Pol) I (PDB ID 2HVI60) and (b) RNA Pol II (PDB ID 2E2H61). DNA Pols, as well as viral RNA Pols, adopt a right‐hand architecture consisting of palm, fingers, and thumb subdomains. Archaeal, bacterial, and eukaryotic RNA Pols have multiple subunits with the catalytic subunits (Rpb1 and Rpb2) and assembly platform (Rpb3‐Rpb11 and Rpb10‐Rpb12) forming the minimal configuration capable of RNA polymerization. Pols may undergo an open → closed conformational transition upon nucleotide binding. The O helix (DNA Pol, A) and trigger loop (RNA Pol, B) are shown in both their open (magenta, PDB IDs 1L3U62 and 1Y1V,63 respectively) and closed (cyan, PDB IDs 2HVI and 2E2H, respectively) conformations. The bridge helix (orange) of RNA Pol does not significantly change in conformation upon nucleotide binding
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Distance between the α‐carbon of a positively charged residue (Arg or Lys) and the two catalytic metals (d1, Å) in crystal structures of ternary Pol/D(R)NA/nucleotide complexes. The positively charged residue is believed to facilitate Watson–Crick base pairing between the incoming nucleotide and templating base. Reprinted with permission from Reference 194 Copyright 2018 American Chemical Society. https://doi.org/10.1021/jacs.7b12446. Further permissions related to the material excerpted should be directed to the American Chemical Society
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(a) Overlap of crystal structures of two‐metal‐ion nucleic‐acid‐processing enzymes. The blue and red spheres represent cations or basic amino residues in similar positions as the two catalytically important potassium ions (K1 and K2) in self‐splicing group II intron ribozymes. (b) Distances (Å) between the K1‐like elements and acidic residues coordinating MA–MB (d K1‐acidic, blue dots) and between K2‐like elements and the substrate (d K2‐substrate, red dots). Reprinted with permission from Reference 137 Copyright 2017 Elsevier Ltd. https://doi.org/10.1016/j.str.2017.11.008. Further permissions related to the material excerpted should be directed to Elsevier Ltd
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(a) Intramolecular H‐bond distance between the 3′‐OH and α‐phosphate groups of the incoming nucleotide (d‐PT, Å) in crystal structures of ternary Pol/D(R)NA/nucleotide complexes. (b) Superimposed crystal structures of ribonucleotides (cyan) and deoxyribonucleotides (white). The intramolecular H‐bond and C3'‐endo sugar conformation are preserved in all structures. Reprinted with permission from Reference 91 Copyright 2016 American Chemical Society. https://doi.org/10.1021/jacs.6b05475. Further permissions related to the material excerpted should be directed to the American Chemical Society
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Nucleotide addition cycle of polymerases (Pols). The active site is shown in the center. Multi‐subunit RNA Pols often have an additional conserved acidic residue (gray) coordinated exclusively to the B‐site metal (MB). In the chemical step, the 3′‐OH group of the primer terminus (red) is deprotonated and attacks the Pα atom of the incoming nucleotide (blue)
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Proposed self‐activated mechanism for nucleotidyl transfer catalyzed by polymerases. (a) The deprotonated 3′‐OH group of the primer terminus attacks the Pα atom of the incoming nucleotide. (b) The primer strand has been extended by one nucleotide. The collective variables (r1–r2) and (r3–r4) were used to investigate (c) the subsequent intramolecular proton transfer and translocation by QM/MM metadynamics simulation. (d) The 3′‐OH group of the newly incorporated nucleotide has been deprotonated, translocation has occurred, and pyrophosphate has been released. (e) The enzyme is ready for the binding of the next nucleotide. Reprinted with permission from Reference 91 Copyright 2016 American Chemical Society. https://doi.org/10.1021/jacs.6b05475. Further permissions related to the material excerpted should be directed to the American Chemical Society
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Proposed mechanisms for nucleotidyl transfer catalyzed by polymerases. In the protein‐mediated mechanism, the 3′‐OH group of the primer terminus is deprotonated by a conserved catalytic residue (Asp or Glu). In the water‐mediated and substrate‐assisted mechanism, the 3′‐OH proton is initially transferred to the α‐phosphate of the incoming nucleotide via a water molecule and ultimately relayed to the β‐phosphate prior to pyrophosphate release and translocation. Reprinted with permission from Reference 126 Copyright 2018 American Chemical Society. https://doi.org/10.1021/acscatal.8b03363. Further permissions related to the material excerpted should be directed to the American Chemical Society
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Structure and Mechanism > Computational Biochemistry and Biophysics
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