Error-prone DNA synthesis on click-ligated templates

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Аннотация

Click ligation is a technology of joining DNA fragments based on azide–alkyne cycloaddition. In the most common variant, click ligation introduces a 4-methyl-1,2,3-triazole (trz) group instead of the phosphodiester bond between two adjacent nucleosides. While this linkage is believed to be biocompatible, little is known about the possibility of its recognition by DNA repair systems or its potential for DNA polymerase stalling and miscoding. Here we report that trz linkage is resistant to several human and bacterial endonucleases involved in DNA repair. At the same time, it strongly blocks some DNA polymerases (Pfu, DNA polymerase β) while allowing bypass by others (phage RB69 polymerase, Klenow fragment). All polymerases except for DNA polymerase β showed high frequency of misinsertion at the trz site, incorporating dAMP instead of the next complementary nucleotide. Thus, click ligation may be expected to produce a large amount of errors if used in custom gene synthesis.

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Авторлар туралы

A. Endutkin

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: dzharkov@niboch.nsc.ru
Ресей, Novosibirsk

A. Yakovlev

Institute of Chemical Biology and Fundamental Medicine SB RAS; Novosibirsk State University

Email: dzharkov@niboch.nsc.ru
Ресей, Novosibirsk; Novosibirsk

T. Zharkov

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: dzharkov@niboch.nsc.ru
Ресей, Novosibirsk

V. Golyshev

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: dzharkov@niboch.nsc.ru
Ресей, Novosibirsk

A. Yudkina

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: dzharkov@niboch.nsc.ru
Ресей, Novosibirsk

D. Zharkov

Institute of Chemical Biology and Fundamental Medicine SB RAS; Novosibirsk State University

Хат алмасуға жауапты Автор.
Email: dzharkov@niboch.nsc.ru

Corresponding Member of the RAS

Ресей, Novosibirsk; Novosibirsk

Әдебиет тізімі

  1. Rostovtsev V.V., Green L.G., Fokin V.V., et al. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective ligation of azides and terminal alkynes, Angew. Chem. Int. Ed., 2002, vol. 41, no. 14, pp. 2596–2599.
  2. Tornøe C.W., Christensen C., and Meldal M. Peptidotriazoles on solid phase: [1,2,3]-Triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides, J. Org. Chem. 2002, vol. 67, no. 9. pp. 3057–3064.
  3. Gierlich J., Burley G.A., Gramlich P.M.E., et al. Click chemistry as a reliable method for the high-density postsynthetic functionalization of alkyne-modified DNA, Org. Lett., 2006, vol. 8, no. 17, pp. 3639–3642.
  4. Seela F., and Sirivolu V.R. DNA containing side chains with terminal triple bonds: Base-pair stability and functionalization of alkynylated pyrimidines and 7-deazapurines, Chem. Biodivers., 2006, vol. 3, no. 5. pp. 509–514.
  5. El-Sagheer A.H., Sanzone A.P., Gao R., et al. Biocompatible artificial DNA linker that is read through by DNA polymerases and is functional in Escherichia coli, Proc. Natl Acad. Sci. U.S.A., 2011, vol. 108, no. 28, pp. 11338–11343.
  6. Sanzone A.P., El-Sagheer A.H., Brown T., and Tavassoli A. Assessing the biocompatibility of click-linked DNA in Escherichia coli, Nucleic Acids Res., 2012, vol. 40, no. 20, pp. 10567–10575.
  7. Dallmann A., El-Sagheer A.H., Dehmel L., et al. Structure and dynamics of triazole-linked DNA: Biocompatibility explained, Chemistry, 2011, vol. 17, no. 52, pp. 14714–14717.
  8. El-Sagheer A.H., and Brown T. New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes, Proc. Natl Acad. Sci. U.S.A., 2010, vol. 107, no. 35, pp. 15329–15334.
  9. El-Sagheer A.H., and Brown T. Efficient RNA synthesis by in vitro transcription of a triazole-modified DNA template, Chem. Commun., 2011, vol. 47, no. 44, pp. 12057–12058.
  10. Rothwell P.J., and Waksman G. Structure and mechanism of DNA polymerases, Adv. Protein Chem., 2005, vol. 71, pp. 401–440.
  11. Endutkin A.V., Yudkina A.V., Zharkov T.D., et al. Recognition of a clickable abasic site analog by DNA polymerases and DNA repair enzymes, Int. J. Mol. Sci., 2022, vol. 23, no. 21, 13353.
  12. Ishchenko A.A., Ide H., Ramotar D., et al. α-Anomeric deoxynucleotides, anoxic products of ionizing radiation, are substrates for the endonuclease IV-type AP endonucleases, Biochemistry, 2004, vol. 43, no. 48, pp. 15210–15216.
  13. Du X., Yang Z., Xie G., et al. Molecular basis of the plant ROS1-mediated active DNA demethylation, Nat. Plants, 2023, vol. 9, no. 2, pp. 271–279.
  14. Shen J.-C., Creighton S., Jones P.A., and Goodman M.F. A comparison of the fidelity of copying 5-methylcytosine and cytosine at a defined DNA template site, Nucleic Acids Res., 1992, vol. 20, no. 19, pp. 5119–5125.
  15. Zahn K.E., Averill A., Wallace S.S., and Doublié S. The miscoding potential of 5-hydroxycytosine arises due to template instability in the replicative polymerase active site, Biochemistry, 2011, vol. 50, no. 47, pp. 10350–10358.
  16. Howard M.J., Foley K.G., Shock D.D., et al. Molecular basis for the faithful replication of 5-methylcytosine and its oxidized forms by DNA polymerase β, J. Biol. Chem., 2019, vol. 294, no. 18, pp. 7194–7201.
  17. Taylor J.-S. New structural and mechanistic insight into the A-rule and the instructional and non-instructional behavior of DNA photoproducts and other lesions, Mutat. Res., 2002, vol. 510, no. 1–2, pp. 55–70.
  18. Obeid S., Baccaro A., Welte W., et al. Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase, Proc. Natl Acad. Sci. U.S.A., 2010, vol. 107, no. 50, pp. 21327–21331.
  19. Xia S., Vashishtha A., Bulkley D. et al. Contribution of partial charge interactions and base stacking to the efficiency of primer extension at and beyond abasic sites in DNA, Biochemistry, 2012, vol. 51, no. 24, pp. 4922–4931.
  20. Beard W.A., Shock D.D., Batra V.K. et al. DNA polymerase β substrate specificity: Side chain modulation of the “A-rule”, J. Biol. Chem., 2009, vol. 284, no. 46, pp. 31680–31689.

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Жүктеу
2. Fig. 1. (a) Scheme for obtaining an oligonucleotide with a triazole internucleosidic linkage and its structure compared with the natural phosphodiester bond. (b) Conformation of the trz-linkage compared with the phosphodiester bond according to nuclear magnetic resonance spectroscopy data. The structure of part of an unmodified DNA duplex (Protein Data Bank structure code 2KUZ, carbon atoms colored white) and a duplex with trz-linkage (code 2L8I, carbon atoms colored black) is shown.

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3. Fig. 2. Representative autoradiograph of the gel after electrophoretic separation of cleavage products of a 32P-labeled DNA strand with trz-linkage by endonucleases APE1 and Xth. The reaction was carried out at 37°C for 1 h in a buffer containing 20 mM HEPES–KOH (pH 8.0), 100 mM KCl, 5 mM MgCl2 or CaCl2 or 1 mM EDTA, 1 mM dithiothreitol (DTT), and 100 μg/ml bovine serum albumin. The DNA substrate concentration was 50 nM, enzymes – 1 μM APE1 or 10 nM Xth. Reaction products were separated by electrophoresis in 20% polyacrylamide gel containing 7 M urea and analyzed using Typhoon 9500 radioluminescent scanning system (GE Healthcare, USA). M – mixture of mobility markers 15, 16, and 33 nucleotides long (M15, M16, and M33, Table 1; corresponding bands indicated by arrows); ss – single-stranded DNA substrate (33trz, Table 1); ds – double-stranded DNA substrate (33trz//T2, Table 1).

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4. Fig. 3. Representative autoradiographs of gels after electrophoretic separation of DNA synthesis products by polymerases KF, RBpol, Pol β (a) and Pfu (b) on matrices with trz-linkage. The reaction was carried out at 37°C for 10 min (20 min for Pfu) in a buffer containing 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2 (for KF and RBpol) or 10 mM MgCl2 (for Pol β), 1 mM DTT; or 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100 for Pfu. The concentration of DNA substrate with 32P-labeled primer was 50 nM, dNTP – 500 μM (individual dNTPs or their equimolar mixture), DNA polymerases – 0.1 units/μl (Pfu) or 10 nM (other polymerases). Reaction products were analyzed as described above. P1, P2 – primers. Numbers under the images indicate the intensity of the bands corresponding to the incorporation of at least one nucleotide passing through the trz-linkage (> n), one nucleotide followed by extension (> n+1), and exonucleolytic degradation of the primer (< n).

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