Abstract
DNA polymerases often incorporate non-canonical nucleotide, i.e., ribonucleoside triphosphates into the genomic DNA. Aberrant accumulation of ribonucleotides in the genome causes various cellular abnormalities. Here, we show the possible role of human nucleotide excision repair (NER) and DNA polymerase η (Pol η) in processing of a single ribonucleotide embedded into DNA. We found that the reconstituted NER system can excise the oxidized ribonucleotide on the plasmid DNA. Taken together with the evidence that Pol η accurately bypasses a ribonucleotide, i.e., riboguanosine (rG) or its oxidized derivative (8-oxo-rG) in vitro, we further assessed the mutagenic potential of the embedded ribonucleotide in human cells lacking NER or Pol η. A single rG on the supF reporter gene predominantly induced large deletion mutations. An embedded 8-oxo-rG caused base substitution mutations at the 3′-neighboring base rather than large deletions in wild-type cells. The disruption of XPA, an essential factor for NER, or Pol η leads to the increased mutant frequency of 8-oxo-rG. Furthermore, the frequency of 8-oxo-rG-mediated large deletions was increased by the loss of Pol η, but not XPA. Collectively, our results suggest that base oxidation of the embedded ribonucleotide enables processing of the ribonucleotide via alternative DNA repair and damage tolerance pathways.
Highlights
DNA replication is essential for maintaining genetic information in living organisms
nucleotide excision repair (NER) is involved in the ribonucleotide removal from DNA, we analyzed whether the human cell-free NER system reconstituted with purified protein factors possesses the ribonucleotide excision activity against a single rG or 8-oxo-rG in the plasmid DNA (Fig. 1a)
Because the efficiency of NER in excising the ribonucleotide was lower compared with the canonical substrate for NER, i.e., UV-induced 6–4 photoproducts (Fig. 1b) (Supplementary Fig. S1), we further assessed the cellular impact of the NER deficiency on the mutagenic potential of an embedded ribonucleotide
Summary
DNA replication is essential for maintaining genetic information in living organisms. Ribonucleotides embedded into DNA are primarily repaired by RNase H2-initiated ribonucleotide excision repair (RER)[8]. Another study has suggested that a ribonucleotide embedded into DNA is a poor substrate for NER function in vitro in both human and Escherichia coli (E. coli)[25]. Based on these findings, the involvement of NER in ribonucleotide removal in cells remains controversial. The presence of 8-oxo-rG in DNA strongly hinders the primer extension reaction catalyzed by human replicative Pol α21. It is of interest to investigate whether TLS Pols exert protective effects against the mutagenicity of incorporated ribonucleotides in cells
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