PIF1: a DNA helicase in yeast mitochondria

• Published in: The EMBO journal Vol. 10; no. 4; pp. 997 - 1007
• Main Authors: Lahaye, A., Stahl, H., Thines‐Sempoux, D., Foury, F.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.04.1991
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Biological and medical sciences; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Repair; DNA, Mitochondrial - genetics; Fundamental and applied biological sciences. Psychology; Gene expression; Genes, Fungal; Kinetics; Microscopy, Immunoelectron; Mitochondria - enzymology; Mitochondria - ultrastructure; Molecular and cellular biology; Molecular genetics; Plasmids; Recombination, Genetic; Restriction Mapping; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Microorganism growth; Recombination; Immunogold labelling; Yeast; Purification; Gene expression; Biological activity; Optimization; Fungi; DNA-dependent ATPase; Mitochondria; Enzymatic activity; Immunoelectron microscopy; Ascomycetes; Localization; Saccharomyces cerevisiae; Hybrid gene; Thallophyta; Unwinding enzyme
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1991.tb08034.x

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix‐localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single‐stranded (ss) DNA‐dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5′ to 3′ direction with respect to the ss DNA on which it binds first.