Identification of a novel human mitochondrial endo-/exonuclease Ddk1/c20orf72 necessary for maintenance of proper 7S DNA levels

Abstract

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.

Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases

“…MGME1 is a 37 kDa monomeric protein that is a mitochondrially-located, metal-dependent exonuclease (Szczesny et al, 2013). MGME1 belongs to the PD-(D/E)XK nuclease superfamily and is a close homologue of the bacterial RecB nuclease (Kornblum et al, 2013; Szczesny et al, 2013).…”

“…MGME1 belongs to the PD-(D/E)XK nuclease superfamily and is a close homologue of the bacterial RecB nuclease (Kornblum et al, 2013; Szczesny et al, 2013). MGME1 selectively degrades ssDNA in both 3′ → 5′ and 5′ → 3′ directions, preferring the latter (Kornblum et al, 2013; Szczesny et al, 2013). It can degrade DNA flap and RNA-DNA flaps (Kornblum et al, 2013; Szczesny et al, 2013), but not circular DNA (Kornblum et al, 2013).…”

“…MGME1 selectively degrades ssDNA in both 3′ → 5′ and 5′ → 3′ directions, preferring the latter (Kornblum et al, 2013; Szczesny et al, 2013). It can degrade DNA flap and RNA-DNA flaps (Kornblum et al, 2013; Szczesny et al, 2013), but not circular DNA (Kornblum et al, 2013). HeLa cells and patient fibroblasts with MGME1 knocked-down have increased amounts and lengths of 7S mtDNA (Nicholls et al, 2014).…”

Mitochondrial Nucleases ENDOG and EXOG Participate in Mitochondrial DNA Depletion Initiated by Herpes Simplex Virus 1 UL12.5

“…Mammalian mitochondria contain a small number of endonucleases, including ENDOG (38,39), EXOG (27), apurinic-apyrimidinic endonucleases 1 and 2 (APEX1/2) (40-42), flap endonuclease 1 (FEN1) (43,44), DNA replication ATP-dependent helicase/nuclease (DNA2) (45), and the recently described endo/exonuclease Ddk1 (46). Of these, APEX1/2, FEN1, and DNA2 seemed unlikely to be involved in mtDNA loss provoked by UL12.5 since they act on specialized DNA substrates, while Ddk1 had not been discovered at the onset of our research.…”

Animal Mitochondrial DNA Replication

“…If so, a high rate of initiation events from O H rarely results in synthesis through the TAS site. The terminated DNA is likely removed by the mitochondrial ssDNA nuclease MGME1 [133, 134]; MGME1 knockdown in cultured cells or MGME1 deficiency in human patients results in a large accumulation of 7S DNA [133, 134]. Pol γ appears to bind preferentially at O H in vivo , and at the site corresponding to the 3′-end of the 7S DNA, which suggests a mechanism for replisome restart from the latter [111].…”