DETERMINING THE SITE AND NATURE OF DNA MUTATIONS WITH THE CLONED MUTYMISMATCH REPAIR ENZYME

The Escherichia coli MutY gene was cloned into a modified pET-11 plasm id which was then transfected into an E.coli HMS174 host for overprodu ction of the MutY mismatch repair (MR) enzyme. Approximately 30-50% of the total cellular protein in the transformed HMS174 cells was isopro pyl-beta-D-thiogalactoside-induced MutY protein, as estimated from the staining intensity on an SDS - PAGE gel following electrophoresis. Th e MutY protein was purified to near homogeneity by cellulose phosphate ion-exchange chromatography followed by gel filtration chromatography . The purified MutY protein had enzyme activities which cleaved the A of a G/A mismatch at the 3' end of the first phosphodiester bond and t hen the 5' end of the second phosphodiester bond of the A. It also cut the A of a C/A mismatch, but to a much lesser extent, and the activit y was DNA sequence-dependent. The reliability of the assay in determin ing the site and nature of a DNA mutation was examined in human tumor DNA samples with known or unknown p53 mutations. In the assay, polymer ase chain reaction-amplified DNA fragments from normal and mutated p53 genes were mixed, denatured and annealed to generate mismatches of G/ A or C/A for cleavage by the MutY MR enzyme, The assay results reveale d the site and nature of known G:C <-> T:A mutations. In addition, a p reviously unknown G:C to T:A mutation, which was misread in the sequen cing analysis of a tumor DNA preparation, was identified by this assay .