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 D...

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Bibliographic Details
Published in:Nucleic acids research 2013-03, Vol.41 (5), p.3144-3161
Main Authors: Szczesny, Roman J, Hejnowicz, Monika S, Steczkiewicz, Kamil, Muszewska, Anna, Borowski, Lukasz S, Ginalski, Krzysztof, Dziembowski, Andrzej
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Catalytic Domain
DNA Cleavage
DNA, Mitochondrial - metabolism
DNA, Single-Stranded - chemistry
Exodeoxyribonucleases - chemistry
Exodeoxyribonucleases - genetics
Exodeoxyribonucleases - metabolism
Gene Expression
Gene Knockdown Techniques
Genome Integrity, Repair and
HeLa Cells
Humans
Hydrogen-Ion Concentration
Mitochondria - enzymology
Mitochondria - genetics
Models, Molecular
Molecular Sequence Annotation
Mutagenesis, Site-Directed
Phylogeny
Protein Structure, Secondary
Protein Transport
RNA, Small Interfering - genetics
Sequence Analysis, DNA
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkt029