TY - JOUR

T1 - Repair and DNA Polymerase Bypass of Clickable Pyrimidine Nucleotides

AU - Endutkin, Anton V.

AU - Yudkina, Anna V.

AU - Zharkov, Timofey D.

AU - Barmatov, Alexander E.

AU - Petrova, Daria V.

AU - Kim, Daria V.

AU - Zharkov, Dmitry O.

N1 - This research was supported by Russian Science Foundation (grant 21-74-10104 to A.V.E., biochemical experiments). Partial salary support from the Russian Ministry of Science and Higher Education is acknowledged (075-15-2022-263).

PY - 2024/6/12

Y1 - 2024/6/12

N2 - Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.

AB - Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.

KW - 5-ethynyl-2′-deoxyuridine

KW - DNA glycosylases

KW - DNA polymerases

KW - DNA repair

KW - click chemistry

KW - metabolic labeling

KW - replication

KW - DNA Repair

KW - Humans

KW - Pyrimidine Nucleotides/chemistry

KW - Click Chemistry

KW - DNA-Directed DNA Polymerase/metabolism

KW - Deoxyuridine/analogs & derivatives

KW - DNA/metabolism

KW - DNA Replication

KW - Uracil-DNA Glycosidase/metabolism

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85197126697&origin=inward&txGid=31aee242b76b8929c8dea67b30b963c6

UR - https://www.mendeley.com/catalogue/e82d8ea8-2c25-38b2-b8e8-9f4fca44d2ed/

UR - https://elibrary.ru/item.asp?id=68154157

U2 - 10.3390/biom14060681

DO - 10.3390/biom14060681

M3 - Article

C2 - 38927084

VL - 14

JO - Biomolecules

JF - Biomolecules

SN - 2218-273X

IS - 6

M1 - 681

ER -