RESUMO
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.
Assuntos
Proteínas de Ligação a DNA/química , Endonucleases Flap/química , Proteínas Fúngicas/química , Mitocôndrias/enzimologia , Podospora/enzimologia , Sequência de Aminoácidos , Bacteriófago M13/metabolismo , Sequência de Bases , Cátions/química , DNA Circular/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Endonucleases Flap/genética , Endonucleases Flap/isolamento & purificação , Endonucleases Flap/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Análise de Sequência de Proteína , Especificidade por SubstratoRESUMO
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.
Assuntos
Endonucleases/isolamento & purificação , Exonucleases/isolamento & purificação , Fungos/enzimologia , Sequência de Aminoácidos , Cátions Bivalentes , Cromatografia em Gel , Reparo do DNA , Eletroforese em Gel de Poliacrilamida , Endonucleases/antagonistas & inibidores , Endonucleases/química , Ativação Enzimática , Exonucleases/antagonistas & inibidores , Exonucleases/química , Magnésio , Mitocôndrias/enzimologia , Peso Molecular , Ribonuclease H/química , Especificidade por Substrato , TemperaturaRESUMO
In Podospora anserina we have described the purification of an endo-exonuclease (Biochim. Biophys. Acta 1574 (1) (2002) 72). Given the description of several nucleases addressed to the mitochondria and known to interact with the PCNA, we sought a possible effect of PCNA on the mt nuclease. A significant stimulation of the nuclease activity with PCNA was observed with double-stranded and flap structure DNA. Immuno-Western blotting experiments realized with monoclonal antibodies raised against the PCNA specifically revealed the presence of a single band of 30 kDa in the mitochondria from the filamentous fungus and yeast. A potential PCNA binding motif was found in the sequences of several mt nucleases and mt proteins involved in the maintenance of the mt DNA with respect to the consensus described by Warbrick. The hypothetical role of the PCNA as a potential regulator of the repair/recombination processes in the maintenance of the mt genome is discussed.