Fluorescence detection of cellular nucleotide excision repair of damaged DNA

Abstract

To maintain genetic integrity, ultraviolet light-induced photoproducts in DNA must be removed by the nucleotide excision repair (NER) pathway, which is initiated by damage recognition and dual incisions of the lesion-containing strand. We intended to detect the dual-incision step of cellular NER, by using a fluorescent probe. A 140-base pair linear duplex containing the (6–4) photoproduct and a fluorophore–quencher pair was prepared first. However, this type of DNA was found to be degraded rapidly by nucleases in cells. Next, a plasmid was used as a scaffold. In this case, the fluorophore and the quencher were attached to the same strand, and we expected that the dual-incision product containing them would be degraded in cells. At 3 h after transfection of HeLa cells with the plasmid-type probes, fluorescence emission was detected at the nuclei by fluorescence microscopy only when the probe contained the (6–4) photoproduct, and the results were confirmed by flow cytometry. Finally, XPA fibroblasts and the same cells expressing the XPA gene were transfected with the photoproduct-containing probe. Although the transfer of the probe into the cells was slow, fluorescence was detected depending on the NER ability of the cells.

Fluorescence detection of DNA mismatch repair in human cells

“…At the beginning of this study, we planned to either incorporate a ribonucleotide 11 , 12 into the mismatch-containing primer used for the preparation of the double-stranded plasmid from the single-stranded DNA, or introduce a nick into the plasmid with a nicking endonuclease 28 . However, we previously found that the plasmids were degraded in the cells within several hours during the culture at 37 °C 23 . Therefore, we decided to test the plasmids without the nick, and expected that a nick would be produced non-specifically in the cells.…”

“…Judging by the results obtained in this study, neither of these two points needed to be considered. It was assumed that the nick was produced by a cellular nuclease, as expected from our previous study 23 , and the replication was avoided by using the pBlueScript vector, which lacks the eukaryotic replication origin.…”

“…In this study, we developed a method to detect the cellular MMR ability by fluorescence. We previously reported the fluorescence-mediated detection of the base excision repair (BER) 22 and nucleotide excision repair (NER) 23 of DNA lesions induced by oxidative stress and UV irradiation, respectively, in cultured human cells (Fig. 1a,b ).…”

“…Fluorescence was detected when the probe was cleaved by NTH1 in human cells. ( b ) Detection of nucleotide excision repair developed in our previous study 23 . A plasmid was used as a scaffold, and the fluorophore and the quencher were attached to the base moieties near a UV-induced lesion.…”

Comparison of Aristolochic acid I derived DNA adduct levels in human renal toxicity models

“…Substantial differences in activity of the involved DNA-repair pathway, i.e. nucleotide excision repair, can be ruled out as both RPTEC/TERT1 (Simon-Friedt et al, 2015) as well as HEK293 (Atanassov et al, 2004, Toga et al, 2014 are NER competent.…”