# Repair and DNA Polymerase Bypass of Clickable Pyrimidine Nucleotides

## TL;DR

This study explores how DNA repair systems and polymerases handle clickable nucleotides, finding that they can be repaired or cause errors, but not at high rates.

## Contribution

The study reveals the repair and bypass mechanisms of bulky clickable pyrimidine nucleotides by DNA glycosylases and polymerases.

## Key findings

- EtU and C8-AlkU are excised by SMUG1 and MBD4 in vitro, but persist longer in human cells than uracil.
- DNA polymerases bypass EtU, C8-AlkU, and C8-AlkC mostly error-free, though some misincorporation occurs, especially with DNA polymerase β.
- Clickable nucleotides may cause mutations, but the frequency of such events in cells is likely low.

## Abstract

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.