HO ENDONUCLEASE CLEAVES MAT DNA IN-VITRO BY AN INEFFICIENT STOICHIOMETRIC REACTION-MECHANISM

Mating type switching in Saccharomyces cerevisiae initiates when Ho en donuclease makes a double-stranded DNA break at the yeast MAT locus. I n this report, we characterize the fundamental biochemical properties of Ho. Using an assay that monitors cleavage of a MAT plasmid, we defi ne an optimal in vitro reaction, showing in particular that the enzyme has a stringent requirement for zinc ions, This suggests that zinc fi nger motifs present in No are important for cleavage, The most unexpec ted feature of Ho, however, is its extreme inefficiency, Maximal cleav age occurs when Ho is present at a concentration of 1 molecule/3 base pairs of substrate DNA. Even under these conditions, complete digestio n requires >2 h. This inefficiency results from two characteristics of Ho, First, Ho recycles slowly from cleaved product to new substrate, in part because the enzyme has an affinity for one end of its double s trand break product. Second, high levels of cleavage in the in vitro r eaction correlate with the appearance of large protein-DNA aggregates, At optimal Ho concentrations, these latter aggregates, referred to as ''florettes,'' have an ordered structure consisting of a densely stai ning central region and loops of radiating DNA, These unusual properti es may indicate that Ho plays a role in other aspects of mating type s witching subsequent to double strand break formation.