Error-prone DNA synthesis on click-ligated templates

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Abstract

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.

About the authors

A. V. Endutkin

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: dzharkov@niboch.nsc.ru
Russian Federation, Novosibirsk

A. O. Yakovlev

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

Email: dzharkov@niboch.nsc.ru
Russian Federation, Novosibirsk; Novosibirsk

T. D. Zharkov

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: dzharkov@niboch.nsc.ru
Russian Federation, Novosibirsk

V. M. Golyshev

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: dzharkov@niboch.nsc.ru
Russian Federation, Novosibirsk

A. V. Yudkina

Institute of Chemical Biology and Fundamental Medicine SB RAS

Email: dzharkov@niboch.nsc.ru
Russian Federation, Novosibirsk

D. O. Zharkov

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

Author for correspondence.
Email: dzharkov@niboch.nsc.ru

Corresponding Member of the RAS

Russian Federation, Novosibirsk; Novosibirsk

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