A DNA Polymerase from Ustilago maydis

Abstract

The 3'' .fwdarw. 5'' DNase activity associated with an U. maydis DNA polymerase hydrolyzed non-complementary 3''-primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyze fully single-stranded polynucleotides, it could hydrolyze such regions less than about 4 nucleotides in length covalently bound to a primer molecule which was base-paired to a complementary template strand. Template-primer combinations containing complementary or non-complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolyzed during the reaction, thus allowing synthesis to occur. No addition of nucleotides onto a conserved non-complementary 3''-primer terminus was detected. The DNase activity therefore fulfilled a proof-reading function during DNA synthesis in vitro.