Identification and biochemical analysis of a mitochondrial endonuclease of Podospora anserina related to curved-DNA binding proteins.
We purified and characterized previously from Podospora anserina mitochondria an endonuclease, active on single-stranded, double-stranded and flap DNA, with RNAse H activity, named P49 according to the major 49 kDa band observed on SDS-PAGE. Edman sequencing allowed us to identify the corresponding gene called nuc49. Here we report the properties of the (His)-tagged NUC49 protein expressed in E. coli. We show that this protein does exhibit an endonuclease activity on plasmid DNA, circular recessed and flap M13 substrate with short protruding single strand. However, in contrast to the mt endonuclease purified fraction it does not present RNase H activity and does not cleave linear flap substrate. The activity differences between the protein expressed in E. coli and the mitochondrial endonuclease fraction previously described are discussed. NUC49 presents a strong homology with the S. pombe CDB4 curved DNA binding protein which belongs to a large family including the human cell cycle protein PA2G4 and is able to bind curved DNA. The results constitute the first description of a mitochondrial endonuclease activity associated to this family of proliferation associated homologous proteins. The function of this endonuclease either in recombination, repair or mt DNA rearrangements remains to be determined.
Laquel-Robert
Patricia
P
CNRS UMR 5097- Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France. patricia.laquel@reger.u-bordeaux2.fr
Sellem
Carole H
CH
Sainsard-Chanet
Annie
A
Castroviejo
Michel
M
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000378:metabolism; D001483:Base Sequence; D002412:Cations / Q000737:chemistry; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D048468:Podospora / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D002412:Cations; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D045585:Flap Endonucleases