Purification and characterization of an endo-exonuclease from Podospora anserina mitochondria.
The senescence phenotype of Podospora anserina wild-type strains depends on mitochondrial (mt) genome stability. Characterization of activities implicated in the maintenance of the mt DNA is therefore essential for a better understanding of these degenerative processes. To address this question we looked for a nuclease activity in this fungal mitochondria. Here we describe the purification of an endo-exonuclease active on single-stranded, double-stranded and flap DNA. The Podospora nuclease also possesses an RNase H activity. Gel filtration chromatography showed a native molecular mass of 90 kDa for the P. anserina enzyme. The highly purified fraction shows a single polypeptide chain of 49 kDa on SDS-PAGE, indicating that the Podospora enzyme is probably active as a dimer. Purification and sequencing of the endolysine digestion peptides of the Podospora mt nuclease suggested that this enzyme could belong to the 5' structure-specific endo-exonuclease family. The possible involvement of this nuclease in mt DNA recombination during the senescence process is evoked.
Bouex
Patrick
P
REGER, UMR 5097, CNRS Université Victor Segalen Bordeaux, France.
Sabourin
Martine
M
Chaignepain
Stephane
S
Castroviejo
Michel
M
Laquel-Robert
Patricia
P
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D002850:Chromatography, Gel; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D004789:Enzyme Activation; D005092:Exonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D005658:Fungi / Q000201:enzymology*; D008274:Magnesium; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D016914:Ribonuclease H / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004720:Endonucleases; D005092:Exonucleases; D016914:Ribonuclease H; C020104:endoexonuclease; D008274:Magnesium