Affinity chromatography of mismatch-specific DNA-glycosylases

Two mismatch-specific DNA-glycosylases MutM (Fpg protein) and MutY are responsible for correction of the mispairs containing oxidatively damaged forms of guanine in DNA. We have developed affinity matrices which have substrate features that permit strong enzyme binding, but almost completely resistant to the cleavage by enzyme. These allow the purification of MutM and MutY glycosylases. Several commercially available, non-silica supports, and three methods of immobilization of the modified oligonucleotide duplex, were compared to establish the best conditions for enzyme purification. The Toyopearl-AF-650M support with the covalent attachment of pure oligonucleotide through an activated 5'-phosphate with further hybridization to a complementary easy changeable modified oligonucleotide, was found to be the best for the affinity chromatography of the MutM and MutY DNA-repair proteins because of its mechanical properties, absence of non-specific binding of proteins and its suitability for modification. This affinity resin can be recycled at least 30 times in the purification of these enzymes to apparent homogeneity from the crude extract of E. coli cells. A correlation was found between the binding affinities of these proteins to the mispair and the concentration of salt which release them from the affinity column. By this approach we were able to show also that the MutY protein of E. coli exists in monomeric and dimeric interconvertible forms having molecular weights of approximately 45 and 85 kDa respectively.