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 endonuclease, responsible for the resolution of recombining mitochondrial DNA molecules in Saccharomyces cerevisiae. We have identified a homologue of Cce1 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 Cce1 act as ligands for the catalytic magnesium ions. Sequence similarities 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 putative role in stabilization of RNA tertiary structure or catalysis of the splicing reaction.
Wardleworth
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BN
Department of Biochemistry, University of Dundee, Dundee DD1 5EH, United Kingdom.
Kvaratskhelia
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White
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MF
D000595:Amino Acid Sequence; D002151:Calorimetry; D002176:Candida albicans / Q000235:genetics*; D020134:Catalytic Domain; D004706:Endodeoxyribonucleases / Q000235:genetics*; D045565:Holliday Junction Resolvases; D008024:Ligands; D008274:Magnesium; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics*; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D020871:RNA Stability; D016601:RNA-Binding Proteins; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D008024:Ligands; C078505:MRS1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; C473486:CCE1 protein, S cerevisiae; D045565:Holliday Junction Resolvases; D008274:Magnesium