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

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 introd...

Full description

Saved in:
Bibliographic Details
Published in:International journal of molecular sciences 2022-11, Vol.23 (21), p.13353
Main Authors: Endutkin, Anton V., Yudkina, Anna V., Zharkov, Timofey D., Kim, Daria V., Zharkov, Dmitry O.
Format: Article
Language:English
Subjects:
Alkynes
AP endonuclease
AP endonucleases
AP site
Base excision repair
Blocking
Chemistry
click chemistry
Crosslinking
Cycloaddition
Deoxyribonucleic acid
DNA
DNA damage
DNA glycosylases
DNA polymerase
DNA repair
DNA-directed DNA polymerase
E coli
Efficiency
Endonuclease
Enzymes
Misreading
Molecular interactions
Phages
Synthetic biology
Tetrahydrofuran
translesion synthesis
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms232113353