TY - JOUR

T1 - Recognition of a Clickable Abasic Site Analog by DNA Polymerases and DNA Repair Enzymes

AU - Endutkin, Anton V.

AU - Yudkina, Anna V.

AU - Zharkov, Timofey D.

AU - Kim, Daria V.

AU - Zharkov, Dmitry O.

N1 - Funding Information: This research was funded by the Russian Science Foundation (Grant No. 21-74-10104). D.V.K. acknowledges support from the Russian Foundation for Basic Research (Grant No. 20-34-90092). D.O.Z. acknowledges partial salary support from the Russian Ministry of Science and Education (Grant No. 121031300056-8). Publisher Copyright: © 2022 by the authors.

PY - 2022/11

Y1 - 2022/11

N2 - Azide–alkyne cycloaddition (“click chemistry”) has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the “A-rule”, directing residual but easily detectable incorporation of dAMP by E. coli DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase β. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and E. coli AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.

AB - Azide–alkyne cycloaddition (“click chemistry”) has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the “A-rule”, directing residual but easily detectable incorporation of dAMP by E. coli DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase β. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and E. coli AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.

KW - AP endonucleases

KW - AP site

KW - click chemistry

KW - DNA glycosylases

KW - DNA polymerases

KW - DNA repair

KW - translesion synthesis

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism

KW - DNA-Directed DNA Polymerase/metabolism

KW - Humans

KW - Escherichia coli/metabolism

KW - DNA Polymerase I/genetics

KW - DNA Repair

KW - DNA Damage

KW - Endonucleases/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85141605485&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/781ef1da-a4f4-36ad-bc7f-5ea5bf226d11/

U2 - 10.3390/ijms232113353

DO - 10.3390/ijms232113353

M3 - Article

C2 - 36362137

AN - SCOPUS:85141605485

VL - 23

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 21

M1 - 13353

ER -