TY - JOUR

T1 - Error-Prone DNA Synthesis on Click-Ligated Templates

AU - Endutkin, A. V.

AU - Yakovlev, A. O.

AU - Zharkov, T. D.

AU - Golyshev, V. M.

AU - Yudkina, A. V.

AU - Zharkov, D. O.

PY - 2024/8/28

Y1 - 2024/8/28

N2 - 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 can be expected to produce a large amount of errors if used in custom gene synthesis.

AB - 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 can be expected to produce a large amount of errors if used in custom gene synthesis.

KW - AP endonucleases

KW - DNA polymerases

KW - DNA repair

KW - click chemistry

KW - mutations

KW - replication

UR - https://www.mendeley.com/catalogue/b488d99b-8606-3788-b6dc-d5d24863c747/

U2 - 10.1134/S1607672924600416

DO - 10.1134/S1607672924600416

M3 - Article

C2 - 39196527

SP - 376

EP - 381

JO - Doklady Biochemistry and Biophysics

JF - Doklady Biochemistry and Biophysics

SN - 1607-6729

ER -