Site-directed mutagenesis of the yeast resolving enzyme Cce1 reveals catalytic residues and relationship with the intron-splicing factor Mrs1

The Holliday junction-resolving enzyme Cce1 is a magnesium-dependent endonu clease, responsible for the resolution of recombining mitochondrial DNA mol ecules in Saccharomyces cerevisiae. We have identified a homologue of Cce1 from Candida albicans and used a multiple sequence alignment to predict res idues important for junction binding and catalysis. Twelve site-directed mu tants have been constructed, expressed, purified, and characterized. Using this approach, we have identified basic residues with putative roles in bot h DNA recognition and catalysis of strand scission and acidic residues that have a purely catalytic role. We have shown directly by isothermal titrati on calorimetry that a group of acidic residues vital for catalytic activity in Cce1 act as ligands for the catalytic magnesium ions. Sequence similari ties between the Cce1 proteins and the group I intron splicing factor Mrs1 suggest the latter may also possess a binding site for magnesium, with a pu tative role in stabilization of RNA tertiary structure or catalysis of the splicing reaction.