# Effects of ribonucleotides on telomeric G4 formation, dynamics, and initiation of ribonucleotide excision repair by RNase H2

## TL;DR

This study shows how ribonucleotides affect the structure and stability of telomeric G4s and hinder their repair by RNase H2.

## Contribution

The paper reveals novel insights into how rNMPs alter G4 conformation and dynamics and reduce RNase H2 cleavage efficiency.

## Key findings

- rNMP substitution in the first dG of a telomeric repeat alters G4 conformation and dynamics.
- rNMPs reduce the thermal stability of G4s depending on their position.
- RNase H2 cleaves rNMPs in G4s less efficiently, especially at less accessible positions.

## Abstract

Human telomeres are composed of TTAGGG repeats that can fold into G-quadruplexes (G4s). G4s can form several different conformations, including parallel, antiparallel 2 + 2 chair, antiparallel 2 + 2 basket, and 3 + 1 parallel/antiparallel. Telomeres are composed of deoxyribonucleotide monophosphates; however, telomerase has been shown to insert ribonucleotide monophosphates (rNMPs) as efficiently as replicative DNA polymerases. Non-telomeric rNMP insertions are deleterious, but the effect on telomeres remains under explored. We systematically investigated 16 variants of the G4-forming telomeric sequence (TTAGGG)4 containing a single rNMP substitution. We generally found that rNMP substitution of the first dG in a repeat (TTAGGG)4 altered the G4 conformation. Incorporation of a rNMP also perturbed G4 folding dynamics, decreasing the population of stably folded molecules and promoting rapid structural transitions. Depending on the rNMP position, we further observed a reduction in overall thermal stability. Additionally, RNase H2, the initiator of ribonucleotide excision repair, had reduced cleavage of rNMPs in G4s, and could only cleave rNMPs at more accessible positions within the G4. Cumulatively, we show that the insertion of rNMPs in telomeric sequences alters the conformation and stability of G4s. This could lead to deleterious effects on telomeric integrity, and these changes may persist due to the difficulty of repairing rNMPs within G4s.

Graphical Abstract