Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro.
The mitochondrial transcription machinery synthesizes the RNA primers required for initiation of leading-strand DNA synthesis in mammalian mitochondria. RNA primers are also required for initiation of lagging-strand DNA synthesis, but the responsible enzyme has so far remained elusive. Here, we present a series of observations that suggests that mitochondrial RNA polymerase (POLRMT) can act as lagging-strand primase in mammalian cells. POLRMT is highly processive on double-stranded DNA, but synthesizes RNA primers with a length of 25 to 75 nt on a single-stranded template. The short RNA primers synthesized by POLRMT are used by the mitochondrial DNA polymerase gamma to initiate DNA synthesis in vitro. Addition of mitochondrial single-stranded DNA binding protein (mtSSB) reduces overall levels of primer synthesis, but stimulates primer-dependent DNA synthesis. Furthermore, when combined, POLRMT, DNA polymerase gamma, the DNA helicase TWINKLE, and mtSSB are capable of simultaneous leading- and lagging-strand DNA synthesis in vitro. Based on our observations, we suggest that POLRMT is the lagging-strand primase in mammalian mitochondria.
Wanrooij
Sjoerd
S
Division of Metabolic Diseases, Karolinska Institutet, Novum, SE-141 86 Stockholm, Sweden.
Fusté
Javier Miralles
JM
Farge
Géraldine
G
Shi
Yonghong
Y
Gustafsson
Claes M
CM
Falkenberg
Maria
M
D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D000074002:DNA Polymerase gamma; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D054629:Genome, Mitochondrial / Q000502:physiology; D006801:Humans; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry
D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C034477:RNA primers; D012313:RNA; D019915:DNA Primase; D012321:DNA-Directed RNA Polymerases; C519567:POLRMT protein, human; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases; C431576:TWNK protein, human