Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation.
Single-stranded DNA binding protein (SSB) plays important roles in DNA replication, recombination and repair through binding to single-stranded DNA. The mammalian mitochondrial SSB (mtSSB) is a bacterial type SSB. In vitro, mtSSB was shown to stimulate the activity of the mitochondrial replicative DNA helicase and polymerase, but its in vivo function has not been investigated in detail. Here we studied the role of mtSSB in the maintenance of mitochondrial DNA (mtDNA) in cultured human cells. RNA interference of mtSSB expression in HeLa cells resulted in rapid reduction of the protein and a gradual decline of mtDNA copy number. The rate of mtDNA synthesis showed a moderate decrease upon mtSSB knockdown in HeLa cells. These results confirmed the requirement of mtSSB for mtDNA replication. Many molecules of mammalian mtDNA hold a short third strand, so-called 7S DNA, whose regulation is poorly understood. In contrast to the gradual decrease of mtDNA copy number, 7S DNA was severely reduced upon mtSSB knockdown in HeLa cells. Further, 7S DNA synthesis was significantly affected by mtSSB knockdown in an oseteosarcoma cell line. These data together suggest that mtSSB plays an important role in the maintenance of 7S DNA alongside its role in mtDNA replication. In addition, live-cell staining of mtDNA did not imply alteration in the organisation of mitochondrial nucleoid protein-mtDNA complexes upon mtSSB knockdown in HeLa cells. This result suggests that the presence of 7S DNA is not crucial for the organisation of mitochondrial nucleoids.
Ruhanen
Heini
H
The Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, UK.
Borrie
Sarah
S
Szabadkai
Gyorgy
G
Tyynismaa
Henna
H
Jones
Aleck W E
AW
Kang
Dongchon
D
Taanman
Jan-Willem
JW
Yasukawa
Takehiro
T
D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D034622:RNA Interference
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D004247:DNA