Analysis of Ribonucleotide Removal from DNA by Human Nucleotide Excision Repair

Abstract

Ribonucleotides are incorporated into the genome during DNA replication. The enzyme RNase H2 plays a critical role in targeting the removal of these ribonucleotides from DNA, and defects in RNase H2 activity are associated with both genomic instability and the human autoimmune/inflammatory disorder Aicardi-Goutières syndrome. Whether additional general DNA repair mechanisms contribute to ribonucleotide removal from DNA in human cells is not known. Because of its ability to act on a wide variety of substrates, we examined a potential role for canonical nucleotide excision repair in the removal of ribonucleotides from DNA. However, using highly sensitive dual incision/excision assays, we find that ribonucleotides are not efficiently targeted by the human nucleotide excision repair system in vitro or in cultured human cells. These results suggest that nucleotide excision repair is unlikely to play a major role in the cellular response to ribonucleotide incorporation in genomic DNA in human cells.

Processing ribonucleotides incorporated during eukaryotic DNA replication

“…However, ribonucleotides in DNA are a poor substrate for the human NER system in vitro and ribonucleotides are not efficiently removed by NER in human cells 65 . It remains to be determined whether NER is involved in repair of genomic ribonucleotides in other eukaryotic systems.…”

Unveiling the non-repair face of the Base Excision Repair pathway in RNA processing: A missing link between DNA repair and gene expression?

“…In the absence of RNase H2, Topoisomerase I cleavage [126] followed by nick processing by Srs2-Exo1 can remove some rNMPs [127,128]. Paired and mispaired rNMPs in DNA can also be targeted by the nucleotide excision repair (NER) in bacteria [129] but likely not in mammalian cells [130], and by the mismatch repair (MMR) systems [131,132]. About this last pathway, it was hypothesized the incorporation of ribonucleotides into mismatching damaged DNA could be interpreted as a putative "benefic" role, verifiable in a signal for the activation of MMR pathway [133].…”

Detection of the Excised, Damage‐containing Oligonucleotide Products of Nucleotide Excision Repair in Human Cells

“…Moreover, using oligonucleotide standards, the in vivo excision assay can be utilized in a quantitative manner to study excision repair activity and kinetics (29). The assay has also provided a mechanism for characterizing the process of nucleotide excision repair in previously unstudied organisms (31) and on novel potential substrates in genomic DNA in vivo (32). Furthermore, taking advantage of the association of excised oligonucleotides with TFIIH and next generation sequencing, the development of the XR-seq method has provided detailed maps of nucleotide excision repair events across the genome in UV-irradiated cells (33, 34).…”