Identification of a novel human mitochondrial endo-/exonuclease Ddk1/c20orf72 necessary for maintenance of proper 7S DNA levels.
Although the human mitochondrial genome has been investigated for several decades, the proteins responsible for its replication and expression, especially nucleolytic enzymes, are poorly described. Here, we characterized a novel putative PD-(D/E)XK nuclease encoded by the human C20orf72 gene named Ddk1 for its predicted catalytic residues. We show that Ddk1 is a mitochondrially localized metal-dependent DNase lacking detectable ribonuclease activity. Ddk1 degrades DNA mainly in a 3'-5' direction with a strong preference for single-stranded DNA. Interestingly, Ddk1 requires free ends for its activity and does not degrade circular substrates. In addition, when a chimeric RNA-DNA substrate is provided, Ddk1 can slide over the RNA fragment and digest DNA endonucleolytically. Although the levels of the mitochondrial DNA are unchanged on RNAi-mediated depletion of Ddk1, the mitochondrial single-stranded DNA molecule (7S DNA) accumulates. On the other hand, overexperssion of Ddk1 decreases the levels of 7S DNA, suggesting an important role of the protein in 7S DNA regulation. We propose a structural model of Ddk1 and discuss its similarity to other PD-(D/E)XK superfamily members.
Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Pawinskiego 5a, 02-106 Warsaw, Poland.
Roman J
RJ
Szczesny
Monika S
MS
Hejnowicz
Kamil
K
Steczkiewicz
Anna
A
Muszewska
Lukasz S
LS
Borowski
Krzysztof
K
Ginalski
Andrzej
A
Dziembowski
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D053837:DNA Cleavage; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017433:Protein Structure, Secondary; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D017422:Sequence Analysis, DNA
D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D034741:RNA, Small Interfering; D005090:Exodeoxyribonucleases; C579164:MGME1 protein, human