Fluorescence detection of DNA mismatch repair in human cells View Full Text


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Article Info

DATE

2018-08-15

AUTHORS

Shunsuke Ito, Miyako Shiraishi, Kazuki Tsuchihashi, Reine Takatsuka, Junpei Yamamoto, Isao Kuraoka, Shigenori Iwai

ABSTRACT

Mismatched base pairs, produced by nucleotide misincorporation by DNA polymerase, are repaired by the mismatch repair (MMR) pathway to maintain genetic integrity. We have developed a method for the fluorescence detection of the cellular MMR ability. A mismatch, which would generate a stop codon in the mRNA transcript unless it was repaired, was introduced into the gene encoding the enhanced green fluorescent protein (EGFP) in an expression plasmid. When MMR-proficient HeLa cells were transformed with this plasmid, the production of active EGFP was observed by fluorescence microscopy. It was assumed that the nick required to initiate the MMR pathway was produced non-specifically in the cells. In contrast, fluorescence was not detected for three types of MMR-deficient cells, LoVo, HCT116, and DLD-1, transformed with the same plasmid. In addition, the expression of a red fluorescent protein gene was utilized to avoid false-negative results. This simple fluorescence method may improve the detection of repair defects, as a biomarker for cancer diagnosis and therapy. More... »

PAGES

12181

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/s41598-018-30733-x

DOI

http://dx.doi.org/10.1038/s41598-018-30733-x

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1106088197

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/30111891


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