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

Abstract

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