The mitochondrial single-stranded DNA binding protein is essential for initiation of mtDNA replication

Abstract

We report a role for the mitochondrial single-stranded DNA binding protein (mtSSB) in regulating mitochondrial DNA (mtDNA) replication initiation in mammalian mitochondria. Transcription from the light-strand promoter (LSP) is required both for gene expression and for generating the RNA primers needed for initiation of mtDNA synthesis. In the absence of mtSSB, transcription from LSP is strongly up-regulated, but no replication primers are formed. Using deep sequencing in a mouse knockout model and biochemical reconstitution experiments with pure proteins, we find that mtSSB is necessary to restrict transcription initiation to optimize RNA primer formation at both origins of mtDNA replication. Last, we show that human pathological versions of mtSSB causing severe mitochondrial disease cannot efficiently support primer formation and initiation of mtDNA replication.

High levels of TFAM repress mammalian mitochondrial DNA transcription in vivo

“…Transient strand-separation of mtDNA may result from mtDNA replication or supercoiling tension, but typically, ssDNA is covered by the mitochondrial single-stranded binding protein (mtSSB) and therefore not accessible to binding by POLRMT and initiation of unspecific transcription (Fusté et al, 2010) (Fig 6). In line with this, there is a drastic increase in unspecific transcription initiation of mtDNA in vivo in a conditional mouse knockout model lacking mtSSB in heart (Jiang et al, 2021). The promoter-specific initiation of mtDNA transcription is thus dependent on both TFAM and mtSSB.…”

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

“…At least two hundred nucleus-encoded proteins are needed for maintenance, replication and transcription of mtDNA, as well as biogenesis of mitochondrial ribosomes [ 4 ]. The basic components of the mtDNA replication machinery are known and mutations in the catalytic and accessory subunits of mitochondrial DNA polymerase ( POLγA and POLγB ) [ 5 , 6 ] the replicative DNA helicase ( TWNK ) [ 7 ], the mitochondrial single-stranded DNA binding protein ( SSBP1 ) [ 8 ] and the mitochondrial genome and maintenance exonuclease 1 ( MGME1 ) [ 9 , 10 ], cause mutations and/or depletion of mtDNA, which, in turn, impair mitochondrial function and cause mitochondrial disease syndromes. According to one model supported by biochemical data, MGME1 is a mitochondrial nuclease that processes newly replicated 5’ DNA ends to facilitate ligation when mtDNA synthesis is completed [ 9 , 11 – 13 ].…”

The Role of Mitochondria in Optic Atrophy With Autosomal Inheritance

“…Many mouse models of autosomal OA have been reported and several common features are apparent from these studies ( Table 2 ). Dominantly inherited autosomal OA-causing mutations are generally found to be embryonic lethal in homozygous mice and as such, many mouse studies linked to autosomal OA are conducted in heterozygotes ( Wakabayashi et al, 2009 ; Jiang et al, 2021 ). Some studies report a progressive loss of visual acuity ( Davies et al, 2007 ; Zaninello et al, 2020 ), although most do not investigate the presence of a visual phenotype.…”

“…Despite many autosomal OA-type phenotypes, there are several phenotypes which are consistently observed in mouse autosomal OA-models which are not generally associated with the corresponding human pathogenic mutations. Most commonly, mice with loss of autosomal OA-causing genes display: reduced body size and difficulty gaining weight ( Smith et al, 2012 ; Wells et al, 2012 ) and cardiomyopathy ( Davies et al, 2008 ; Jiang et al, 2021 ). In many cases, these symptoms are not detectable in patients carrying the comparable pathogenic mutation and whilst many autosomal OA-causing genes are thought to have a developmental role in humans, there is presently little evidence for developmental abnormalities affecting these organs in patients ( Maltecca et al, 2008 ; Caglayan et al, 2020 ).…”

“…Summary of phenotypes observed in in vivo models of autosomalOA. et al, 2008), Ssbp1(Jiang et al, 2021), Dnm1l(Wakabayashi et al, 2009) Wfs1(Cairns, 2019) Opa1(Shahrestani et al, 2009), Dnm1l(Batzir et al, 2019), Ssbp1(Maier et al, 2001), Afg3l2(Pareek and Pallanck, 2020) Aco2(Simmer et al, 2003), Afg3l2(Zubovych et al, 2010), Dnm1l(Breckenridge et al, 2008) Reduced lifespanOpa3(Davies et al, 2008), Afg3l2(Maltecca et al, 2008), Mcat (Smith et al, 2012) Opa1 (Rahn et al, 2013), Opa3 (Pei et al, 2010) Opa1 (Tang et al, 2009), Wfs1 (Sakakibara et al, 2018), Afg3l2 (Pareek and Pallanck, 2020) Opa1 (Byrne et al, 2019) Abnormal mitochondria Opa1 (Davies et al, 2008), Sspb1 (Jiang et al, 2021), Dnm1l (Gerber et al, 2017b), Opa3 (Powell et al, 2011), Afg3l2 (Mancini et al, 2019), Mcat (Smith et al, 2012) Opa1 (Eijkenboom et al, 2019), Wfs1 (Cairns, 2019), Slc25a46 (Wan et al, 2016) Opa1 (Shahrestani et al, 2009), Dnm1l (Altanbyek et al, 2016), Ssbp1 (Maier et al, 2001), Afg3l2 (Pareek and Pallanck, 2020), Slc25a46 (Ali et al, 2020) Opa1 (Kanazawa et al, 2008), Ssbp1 (Sugimoto et al, 2008), Afg3l2 (Benedetti et al, 2006), Rtn4ip1 (Fujii et al, 2011), Dnm1l (Scholtes et al, 2018)Reduced visual acuityOpa1(Zaninello et al, 2020), Opa3 (Davies et al, 2008), Wfs1 (Bonnet-Wersinger et al, 2014) et al, 2012), Opa3 (Davies et al, 2008), Afg3l2 (Maltecca et al, 2009), Wfs1 (Waszczykowska et al, 2020) Opa3 (Pei et al, 2010), Wfs1 (Cairns, 2019), Slc25a46 (Abrams et al, 2015) Opa1 (Shahrestani et al, 2009), Wfs1 (Sakakibara et al, 2018), Afg3l2 (Pareek and Pallanck, 2020) N/A Motor defects Opa1 (Sarzi et al, 2012), Opa3 (Davies et al, 2008), Afg3l2 (Maltecca et al, 2008), Mcat (Smith et al, 2012) Opa1 (Rahn et al, 2013), Opa3 (Pei et al, 2010), Slc25a46 (Abrams et al, 2015) Opa1 (Shahrestani et al, 2009), Wfs1 (Sakakibara et al, 2018), Afg3l2 (Pareek and Pallanck, 2020), Slc25a46 (Suda et al, 2018) Opa1 (Byrne et al, 2019), Dnm1l (Scholtes et al, 2018)…”