Fluorescence detection of cellular nucleotide excision repair of damaged DNA View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2014-07-04

AUTHORS

Tatsuya Toga, Isao Kuraoka, Shun Watanabe, Eiji Nakano, Seiji Takeuchi, Chikako Nishigori, Kaoru Sugasawa, Shigenori Iwai

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. More... »

PAGES

5578

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/srep05578

DOI

http://dx.doi.org/10.1038/srep05578

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1036640533

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24993089


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