Ho endonuclease cleaves MAT DNA in vitro by an inefficient stoichiometric reaction mechanism

Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a double-stranded DNA break at the yeast MAT locus. In this report, we characterize the fundamental biochemical properties of Ho. Using an assay that monitors cleavage of a MAT plasmid, we define an optimal in vitro reaction, showing in particular that the enzyme has a stringent requirement for zinc ions, This suggests that zinc finger motifs present in No are important for cleavage, The most unexpected feature of Ho, however, is its extreme inefficiency, Maximal cleavage occurs when Ho is present at a concentration of 1 molecule/3 base pairs of substrate DNA. Even under these conditions, complete digestion 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 strand break product. Second, high levels of cleavage in the in vitro reaction 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 staining central region and loops of radiating DNA, These unusual properties may indicate that Ho plays a role in other aspects of mating type switching subsequent to double strand break formation.