FIGURE 3.
A nine amino acid Dna2 peptide is sufficient for activation of Mec1. A, titration of the Dna2(1–499) domain or the Dna2-1 peptide (sequence given in B) in the standard Mec1 assay with Rad53-kd as substrate (see “Experimental Proceduress”). B, effect on peptide activity of substituting the Trp-128 residue with other aromatics. C, reduction of the size of the peptide results in gradual loss in affinity. Peptide size numbering is shown in B, and Km values determined. D, residues in the Dna2-1 peptide were mutated to alanine, as indicated in the graph, and their ability to stimulate Mec1 was quantified relative to the wild-type peptide. Peptide concentrations ranged from 1.5–50 μm. Data represent the average of at least three independent experiments for peptides that showed activity, and of two experiments for peptides that were inactive. Error bars are standard error of the mean. Pro-122 and Pro-132 were simultaneously mutated to alanines.