Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication.
Mitochondrial DNA is replicated by a unique enzymatic machinery, which is distinct from the replication apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we can faithfully reconstitute OriL-dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure. At this stage, POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase gamma, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mitochondria.
Fusté
Javier Miralles
JM
Division of Metabolic Diseases, Karolinska Institutet, Novum, SE-141 86 Stockholm, Sweden.
Wanrooij
Sjoerd
S
Jemt
Elisabeth
E
Granycome
Caroline E
CE
Cluett
Tricia J
TJ
Shi
Yonghong
Y
Atanassova
Neli
N
Holt
Ian J
IJ
Gustafsson
Claes M
CM
Falkenberg
Maria
M
D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000096:biosynthesis* / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D020868:Gene Silencing; D006801:Humans; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011071:Poly T / Q000737:chemistry; D018741:Replication Origin
D004272:DNA, Mitochondrial; D011071:Poly T; D012321:DNA-Directed RNA Polymerases; C519567:POLRMT protein, human