Graduate Thesis Or Dissertation

 

An assay for screening cells for mismatch repair proficiency in vivo Public Deposited

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  • Mismatch repair is one of the mechanisms by which cells ensure genomic stability. Deficiencies in mismatch repair (MMR) increase mutation rates and cancer risks. In the well-characterized methyl-directed Escherichia co/i system, MMR is initiated by MutS, Mut L, and MutH proteins. The single MutS protein and the single MutL protein in prokaryotes are diverged into several MSH (MutS homolog) proteins and MLH (MutL homolog) proteins in eukaryotes. Several germline mutations in human mismatch repair genes, mainly /iMSH2 and hMLHJ, have been associated with hereditary non-polyposis colorectal cancer (HNPCC). MMRdeficient cells show a higher resistance to some anti-tumor reagents. Early detection of mismatch repair defects might be useful in anti-tumor drug selection. In this study, I wanted to develop an assay for screening MMR-proficient cells. First, I constructed a gapped plasmid, employing the tandem-nick method (Wang & Hays, 2001) and generated G/A base-base mismatched substrates by annealing a synthetic oligomer into the gapped molecules. The plasmid with the incorrect adenine on the template strand encodes a truncated non-functional protein, and the repair of this incorrect adenine to the correct cytosine would produce an active enzyme. A strand-specific and site-specific nick site was generated by a DNA single-strand nicking enzyme, N.Bpu 10! endonuclease. This repair-reporter plasmid was transfected to a number of different cells, including lymphoblastoid (TK6 and MT1) cells, mouse fibroblast (mc2 and mc5) cells, and tumor (HCT116) cells. Luciferase activities in cell lysates were assayed to determine the efficiency of correction mismatched G/A to G/C, which encodes an active protein. To normalize transfection efficiencies as well as lysate preparation variations, plasmid pCH1 10, which encodes full-length E. coli -galactosidase, was used as second reporter gene in co-transfection experiments. The apparent repair efficiencies proved to be independent of the mismatch-repair genotype in lymphoblastoid cells and were slighter higher in mismatch-repair-proficient mc5 mouse cells than in mc2 mismatch-repair-deficient cells but were low in general. The results indicate that the G/A base-base mismatch is very likely repaired via another activity. A likely possibility is the hMYH DNA glycosylase, which can cleave adenine from a G/A mismatch as well as from A/8-oxo-guanine. I was able to quantify the following repair efficiencies for a G/A mismatch, in supercoiled DNA: 50% in lymphoblast cells, 5-14% in mouse fibroblast cells, and about 11% in tumor cells. However, I also found that a G/A mispair may not be a good substrate for screening MMR proficiency in vivo.
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