A Pure Dna Mismatch Repair Effect Executing 5-Fluorouracil Cytotoxicity For Colorectal Cancers

Background. Current models have proposed that part of the DNA damage response (DDR) is dependent on the DNA mismatch repair (MMR) system, but there are no good cellular models that demonstrate that MMR proteins recognize DNA damage to subsequently signal cell-cycle checkpoints or apoptosis machinery. For measurement of DNA MMR activity in a living cell, un-nicked circular heteroduplex plasmid DNA is reported to be useful as a substrate. However, multiple factors significantly affect the yield of heteroduplex plasmid DNA, which voids the ability to demonstrate MMR action for DDR. Aims: To establish a method for efficient preparation of a heteroduplex plasmid containing a DNA damage lesion. Methods. We used 5fluorodeoxyuracil (5FdU) as a DNA damage lesion, which models 5-fluorouracil incorporated into DNA as we previously demonstrated for In Vitro MMR binding assay. We first compared the efficacy between two methods of 5FdU-containing heteroduplex plasmid (5FdU plasmid) construction; one was constructed by a ligation reaction between linearized double-strand DNA (dsDNA) plasmid and annealed synthetic oligonucleotide containing 5FdU (ligation method), and the other was by primer extension/ ligation reaction by using both T4 DNA polymerase and T4 DNA ligase at one time after annealing a 5FdU-containing phosphorylated oligomer with a pGEM7Zf(+) single-strand DNA (ssDNA) plasmid (primer extension method). Based on the results, we further analyzed determinant factors of each process to purify high quality 5FdU plasmid. Results. With the ligation method, we yielded less than 1.0 μg from 10 μg of parental dsDNA plasmid, whereas with the primer extension method, 7.4-12 μg could be purified from the 10 μg ssDNA plasmid. We found it easier to detect the heteroduplex site by direct sequencing with the primer extension method compared to the ligation method. To improve quality and quantity of 5FdU plasmid preparation, we realized the following determinant factors: 1) It is easier and more efficient to isolate ssDNA plasmid using M13K07 helper phage through generating individual transformed colonies of JM109/template dsDNA plasmid/M13K07 than conventional M13K07 infection; 2) Phosphorylation of 5FdU-containing oligomer improved the closed 5FU plasmid recovery rate; 3) A shorter time for primer extension/ligation reaction had higher yield due to T4 polymerase having delayed 3'-5' exonuclease activity. The purified 5FdU plasmids could be transfected, and enabled us to observe cellular cytotoxicity when compared to positive and the negative control plasmids, which suggests usefulness for further analysis of DDR response.Conclusion. We established efficient production of a heteroduplex plasmid containing DNA damage lesion, which will be useful to analyze a pure DDR effect in live cells.