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.
Highlights
DNA must be replicated prior to cell division
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)
The mRNA produced under the control of the cytomegalovirus (CMV) promoter in the cells transformed with this plasmid would contain a stop codon, UAG, in the absence of the cellular mismatch repair (MMR) activity
Summary
DNA must be replicated prior to cell division. In eukaryotic cells, three DNA polymerases, α, δ, and ε, are primarily involved in the replication[1]. DNA polymerase α initiates the replication with its primase activity, and DNA polymerases δ and ε synthesize the lagging and leading strands, respectively, by nucleotide polymerization in the 5′ to 3′ direction, depending on the base-pair formation with the template strand. This process must be accurate because the misincorporation of a nucleotide, to form a mismatch, would induce a genetic mutation, which results in carcinogenesis or cell death. In IHC, false-positive results may be obtained for dysfunctional MMR gene products containing a single amino acid substitution, outside the dimerization domains[21]
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