Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases.

Valentina Riva,Simone Sabbioneda,Anna Garbelli,Assunta Gagliardi,Luca Bini,Martina Schroeder,Claudia Immacolata Trivisani,Francesca Arena,Federica Casiraghi,Antonio Maffia,Giovanni Maga

doi:10.1093/nar/gkaa948

Abstract

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5′ of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols β and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.

Highlights

Compelling experimental evidence accumulating in the last years, led to the realization that the most abundant type of endogenous DNA damage in mammalian cells is represented by the >1 million ribonucleotides erroneously incorporated into the genome at each round of replication
It is already known that DDX3X is able to resolve DNA:RNA hybrids and RNA secondary structures and it has been implicated in RNA degradation processes [14]
When incubated in the presence of a specific oligonucleotide dsDNA substrate containing a single rCMP embedded in one strand at a specific position (Substrate *D39R1D15: D55 Figure 1A), recombinant purified human DDX3X (Supplementary Figure S1a and b) cut at the 5 side of the single rNMP, generating a product identical to the one of RNaseH2 (Figure 1A, compare lanes 1–5 with lanes 6– 10), albeit with lower efficiency (Figure 1B)

Summary

Introduction

Compelling experimental evidence accumulating in the last years, led to the realization that the most abundant type of endogenous DNA damage in mammalian cells is represented by the >1 million ribonucleotides (rNMPs) erroneously incorporated into the genome at each round of replication. RNMPs misincorporation leads to deleterious consequences: their presence into the genome makes the sugar-phosphate backbone ∼100 000- times more prone to hydrolysis due to the presence of the 2 -OH in their ribose ring. This might lead to DNA breaks and replication fork arrest.

Methods

Results

Conclusion