Mitochondrial RNA Polymerase Is Needed for Activation of the Origin of Light-Strand DNA Replication

• Published in: Molecular cell Vol. 37; no. 1; pp. 67 - 78
• Main Authors: Fusté, Javier Miralles, Wanrooij, Sjoerd, Jemt, Elisabeth, Granycome, Caroline E., Cluett, Tricia J., Shi, Yonghong, Atanassova, Neli, Holt, Ian J., Gustafsson, Claes M., Falkenberg, Maria
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2010
• Subjects: Annan medicinsk grundvetenskap; biosynthesis; chemistry; DNA; DNA Replication; DNA, Mitochondrial - biosynthesis; DNA, Mitochondrial - chemistry; DNA-Directed RNA Polymerases; DNA-Directed RNA Polymerases - physiology; Gene Silencing; Genetic; Humans; Medicin och hälsovetenskap; Mitochondrial; Models, Genetic; Nucleic Acid Conformation; Other Basic Medicine; physiology; Poly T; Poly T - chemistry; PROTEINS; Replication Origin; PROTEINS; DNA
• ISSN: 1097-2765; 1097-4164; 1097-4164
• DOI: 10.1016/j.molcel.2009.12.021

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 γ, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mitochondria.