DNA hydrolytic activity associated with the Ustilago maydis REC1 gene product analyzed on hairpin oligonucleotide substrates

The REC1 gene of Ustilago maydis functions in the maintenance of genome sta bility as evidenced by the mutator phenotype resulting from inactivation of the gene. The biochemical function of the Rec1 protein was previously iden tified as a 3'-5'-directed DNA exonuclease. Here studies on the mechanism o f action of Red were performed using radiolabeled oligonucleotide DNAs as s ubstrates, enabling detection of single cleavage events after electrophores is on DNA sequencing gels. The oligonucleotides that were utilized were des igned to be self-annealing so that they formed hairpin structures. This sim plified interpretation of the data since each molecule contained only one 3 '-terminus. Analysis revealed that digestion proceeded by a distributive mo de of action and that degradation of DNA was governed by an interplay betwe en sequence context and conformation. The preferential substrate was DNA wi th a recessed 3'-end. It was discovered that the enzyme had abasic endonucl ease activity, was capable of initiating at an internal nick, and had no pr eference for mismatched bases either internally or terminally. Endonucleoly tic cleavage was 5' to the abasic site.