Novel plasmids for the fluorescence-based evaluation of DNA mismatch repair in human cells

Abstract

Mismatch repair (MMR) is a highly conserved DNA repair pathway that corrects mismatched bases during DNA replication. The biological significance of MMR in human cells is underscored by the fact that dysfunction of the MMR pathway results in Lynch syndrome, which is associated with a genetic predisposition to different cancer types. We have previously established a reporter mismatch plasmid to evaluate MMR using fluorescent proteins in living cells. However, the preparation of these plasmids requires significant amounts of time and money, which reduces their broad applicability. To overcome the abovementioned limitations, we produced in this study a novel reporter plasmid, pBSII NLS-MC-EGFP-tdTomato (pBET2), that can be used in the oligo swapping method. In this method, a nicking endonuclease produces a single-stranded DNA gap on a double-stranded DNA plasmid that can be replaced by ligation with synthetic oligonucleotides. It is significantly easier and more user-friendly than previous assays, which require in vitro DNA synthesis with single-stranded plasmid DNA and purification using ultracentrifugation in cesium chloride-ethidium bromide gradients. The plasmid also contains a nicking site that allows the MMR repair machinery to efficiently distinguish the newly synthesized strand as a target for repair. In addition, a nuclear localization signal facilitates green fluorescent protein expression in the nucleus, which helps to verify the effectiveness of MMR using fluorescence microscopy. Similar to the previous reporter plasmid, this construct facilitates the assessment of MMR proficiency in human living cells via the expression of fluorescent proteins while overcoming many of the negative aspects of the previous protocol.