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Structural insights into DNA replication without hydrogen bonds.

Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Romesberg FE, Marx A
J Am Chem Soc 135 18637-43 (2013)
Related entries: 4c8k, 4c8l, 4c8n, 4c8o, 4cch

Cited: 44 times
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Abstract

The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the prechemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchemistry complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.

Articles - 4c8m mentioned but not cited (2)

  1. Structural insights into DNA replication without hydrogen bonds. Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Romesberg FE, Marx A. J Am Chem Soc 135 18637-18643 (2013)
  2. Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II. Oh J, Shin J, Unarta IC, Wang W, Feldman AW, Karadeema RJ, Xu L, Xu J, Chong J, Krishnamurthy R, Huang X, Romesberg FE, Wang D. Nat Chem Biol 17 906-914 (2021)


Reviews citing this publication (11)

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Articles citing this publication (31)

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  5. Structural basis for a six nucleotide genetic alphabet. Georgiadis MM, Singh I, Kellett WF, Hoshika S, Benner SA, Richards NG. J Am Chem Soc 137 6947-6955 (2015)
  6. New codons for efficient production of unnatural proteins in a semisynthetic organism. Fischer EC, Hashimoto K, Zhang Y, Feldman AW, Dien VT, Karadeema RJ, Adhikary R, Ledbetter MP, Krishnamurthy R, Romesberg FE. Nat Chem Biol 16 570-576 (2020)
  7. Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet. Dhami K, Malyshev DA, Ordoukhanian P, Kubelka T, Hocek M, Romesberg FE. Nucleic Acids Res 42 10235-10244 (2014)
  8. Expansion of the Genetic Alphabet: A Chemist's Approach to Synthetic Biology. Feldman AW, Romesberg FE. Acc Chem Res 51 394-403 (2018)
  9. Site-specific enzymatic introduction of a norbornene modified unnatural base into RNA and application in post-transcriptional labeling. Domnick C, Eggert F, Kath-Schorr S. Chem Commun (Camb) 51 8253-8256 (2015)
  10. Supramolecular Control over Split-Luciferase Complementation. Bosmans RP, Briels JM, Milroy LG, de Greef TF, Merkx M, Brunsveld L. Angew Chem Int Ed Engl 55 8899-8903 (2016)
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  12. Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of Alkene- versus Alkyne-Modified Nucleotides. Hottin A, Betz K, Diederichs K, Marx A. Chemistry 23 2109-2118 (2017)
  13. Tautomeric Equilibria of Nucleobases in the Hachimoji Expanded Genetic Alphabet. Eberlein L, Beierlein FR, van Eikema Hommes NJR, Radadiya A, Heil J, Benner SA, Clark T, Kast SM, Richards NGJ. J Chem Theory Comput 16 2766-2777 (2020)
  14. Chemical Stabilization of Unnatural Nucleotide Triphosphates for the in Vivo Expansion of the Genetic Alphabet. Feldman AW, Dien VT, Romesberg FE. J Am Chem Soc 139 2464-2467 (2017)
  15. Snapshots of a modified nucleotide moving through the confines of a DNA polymerase. Kropp HM, Dürr SL, Peter C, Diederichs K, Marx A. Proc Natl Acad Sci U S A 115 9992-9997 (2018)
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