Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro Sjoerd Wanrooij*†, Javier Miralles Fuste´ *, Ge´ raldine Farge*, Yonghong Shi*†, Claes M. Gustafsson*†‡, and Maria Falkenberg*†‡ *Division of Metabolic Diseases, Karolinska Institutet, Novum, SE-141 86 Stockholm, Sweden; and †Department of Medical Biochemistry and Cell Biology, Go¨teborg University, Box 440, SE-405 30 Go¨teborg, Sweden Communicated by I. Robert Lehman, Stanford University School of Medicine, Stanford, CA, June 4, 2008 (received for review March 14, 2008) The mitochondrial transcription machinery synthesizes the RNA strand promoters (LSP) generates transcripts of almost genomic primers required for initiation of leading-strand DNA synthesis in length, which are processed to yield the individual mRNA and mammalian mitochondria. RNA primers are also required for ini- tRNA molecules (10, 11). LSP-dependent transcription also tiation of lagging-strand DNA synthesis, but the responsible en- generates the primers needed at OH (12), and RNA attached to zyme has so far remained elusive. Here, we present a series of the newly synthesized H strand has been detected in both mouse observations that suggests that mitochondrial RNA polymerase and human cells (13). Primer formation may include an RNase (POLRMT) can act as lagging-strand primase in mammalian cells. MRP-dependent cleavage of the transcribed L-strand RNA at POLRMT is highly processive on double-stranded DNA, but synthe- the initiation sites of DNA replication (14). Alternatively, spe- sizes RNA primers with a length of 25 to 75 nt on a single-stranded cific mtDNA sequences may direct premature transcription template. The short RNA primers synthesized by POLRMT are used termination and primer formation near OH (15). by the mitochondrial DNA polymerase ␥ to initiate DNA synthesis As described above, both the modified strand-asymmetric model in vitro. Addition of mitochondrial single-stranded DNA binding and the strand-coupled model for mtDNA replication require protein (mtSSB) reduces overall levels of primer synthesis, but initiation of lagging-strand DNA synthesis at multiple sites. How- stimulates primer-dependent DNA synthesis. Furthermore, when ever, the enzyme required for lagging-strand primer synthesis has combined, POLRMT, DNA polymerase ␥, the DNA helicase TWIN- not yet been identified. Bioinformatic and biochemical analysis of KLE, and mtSSB are capable of simultaneous leading- and lagging- the mitochondrial DNA helicase, TWINKLE, suggest that this strand DNA synthesis in vitro. Based on our observations, we protein also harbors a primase activity in at least some nonmeta- suggest that POLRMT is the lagging-strand primase in mammalian zoan cells (16–18), but the primase activity has been lost in mitochondria. mammalian TWINKLE (17, 19). There have also been reports of an OL-specific primase activity in mitochondrial extracts, but the DNA replication ͉ mitochondrion ͉ primase corresponding protein has not been identified (20). In this report, we characterize mitochondrial RNA polymer- uman mitochondria have a small double-stranded DNA ase (POLRMT) and provide biochemical evidence suggesting H(dsDNA) genome of 16.6 kb, which contains two origins of that this polymerase is the lagging-strand primase in mammalian replication, OH and OL (1). According to the strand- mitochondria. Our investigations were initially prompted by a displacement model for mitochondrial DNA (mtDNA) replica- previous report, which identified a primase activity in nuclear tion, DNA synthesis is continuous on both strands and takes extracts of herpes simplex virus type 1-infected Vero cells (21). place in a strand-asymmetric mode. DNA synthesis from OH is The observed primase synthesized short oligoribonucleotides, unidirectional and proceeds to displace the parental heavy with the predominant products in the range of 30 to 60 nt. Based strand (H-strand). After leading-strand synthesis has reached on the apparent molecular weight and the enzymatic character- two-thirds of the genome, it activates OL and lagging-strand istics of the observed activity, the authors concluded that the DNA synthesis then initiates in the opposite direction (2–4). responsible enzyme most likely was POLRMT, but the enzyme Recently, analysis of mtDNA replication intermediates by was never directly identified. The biochemical analysis presented atomic force microscopy revealed that the strand-displacement here supports this previous report and demonstrates that model is an oversimplification and that additional initiation sites POLRMT has two distinct modes of action. The enzyme effi- for lagging-strand mtDNA synthesis exist on the light strand ciently transcribes long regions of dsDNA, but becomes much (L-strand) (5). However, individually, these multiple initiation less processive on single-stranded DNA (ssDNA), producing sites may be less abundant, explaining why only OL was identified only short RNA molecules of 25–75 nt. The short RNA primers when 5Ј-end mapping was used to identify initiation sites for can be used by the mitochondrial POL␥ to initiate DNA lagging-strand mtDNA synthesis (3). synthesis in vitro and this reaction is stimulated by mitochondrial The strand-asymmetric model for mtDNA replication has ssDNA binding protein (mtSSB). We have demonstrated that been challenged by two dimensional neutral/neutral agarose gel the minimal mtDNA replisome (POL␥, mtSSB, and TWINKLE) electrophoresis (2DNAGE), demonstrating the presence of con- can support leading-strand DNA synthesis on a double-stranded ventional duplex mtDNA replication intermediates, indicative of DNA template (22). We now find that addition of POLRMT to coupled leading and lagging-strand DNA synthesis (6, 7). One this system, leads to simultaneous leading- and lagging-strand specific class of replication intermediates identified by 2DNAGE DNA synthesis in vitro. incorporates RNA on the lagging strand across the entire genome (8). The idea has been put forward that the lagging Author contributions: S.W., J.M.F., C.M.G., and M.F. designed the research; S.W., J.M.F., strand is initially laid down as RNA and subsequently replaced G.F., Y.S., and M.F. performed the research; Y.S. contributed new reagents/analytic tools; by DNA in a maturation step involving the mitochondrial DNA S.W., J.M.F., G.F., C.M.G., and M.F. analyzed data; S.W., J.M.F., C.M.G., and M.F. wrote the polymerase (POL␥), but the exact mechanisms remain to be article. elucidated (8, 9). The authors declare no conflict of interest. The mitochondrial transcription machinery synthesizes the ‡To whom correspondence may be addressed. E-mail: [email protected] or RNA primers required for initiation of leading-strand DNA [email protected]. synthesis at OH. Transcription from the mitochondrial light © 2008 by The National Academy of Sciences of the USA 11122–11127 ͉ PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805399105 Downloaded by guest on September 24, 2021 A B 0.1 M NaCl 1.2 M NaCl t Fractions: 1 5 9 13 17 21 25 29 32 34 36 38 40 43 47 51 55 (kD) 150 pBluescripssDNA M13mpssDNA18 100 POLRMT - + - + 75 50 (nts) 100 (nts) 75 100 50 50 25 25 Fractions: 1 5 9 13 17 21 25 29 32 34 36 38 40 43 47 51 55 Fig. 1. POLRMT synthesizes short RNA stretches on ssDNA templates. RNA synthesis by POLRMT was monitored as described in Material and Methods by labeling the RNA. (A) RNA synthesis by POLRMT (500 fmol) was carried out on pBluescript or M13mp18 ssDNA templates and the products were separated on a denaturing polyacrylamide gel (10%). (B) POLRMT peak fractions (10 ␮l) from purification over MonoS were separated by SDS/PAGE (10%) and revealed with Coomassie Brilliant Blue staining (Upper). The RNA synthesis of the protein fractions (5 ␮l) in the upper panel was analyzed in the presence of M13mp18 ssDNA (Lower). The beginning and the end of the linear salt gradient are indicated. Results POL␥ Can Use POLRMT-Synthesized Primers to Initiate DNA Replica- POLRMT Efficiently Synthesizes Short RNA Fragments on an ssDNA tion in Vitro. POL␥ can initiate DNA synthesis from a prean- Template. A previous report suggested that POLRMT possesses nealed primer on M13mp18 ssDNA (Fig. 3A, lane 1). In the primase activity (21) and we decided to experimentally address presence of this template POL␥ incorporates dNTPs into DNA this possibility. First, we analyzed the polymerase activity of at a rate of approximately 120 fmol/min. In the absence of a recombinant POLRMT on ssDNA (Fig. 1A). We observed that primer, POL␥ is unable to initiate DNA synthesis on M13mp18 POLRMT synthesizes relatively short RNA species on ssDNA ssDNA (Fig. 3A, lane 3). However, in the presence of POLRMT, (25–75 ribonucleotides), confirming previous results with par- POL␥ can efficiently incorporate dNTPs into DNA (Fig. 3A, tially purified native POLRMT (21). The RNA primers were lanes 4–7), suggesting that POL␥ utilizes RNA primers synthe- strongly reduced (Ͼ95%) after RNase H treatment, indicating sized by POLRMT to initiate DNA synthesis. POLRMT can that the RNA primers were annealed to ssDNA (data not synthesize multiple primers on each M13mp18 ssDNA molecule shown). and higher levels of DNA synthesis are therefore observed in the Even though we estimated the purity of our recombinant presence of POLRMT (Fig. 3A, lanes 4–7), compared with the POLRMT preparation to be Ͼ95%, we decided to follow the preannealed primer template (Fig. 3A, lane 1), which only activity during another step of purification to exclude the contains one possible initiation site for DNA synthesis. possibility that the short RNA primers were formed by a To investigate the destiny of RNA primers during DNA contaminant primase from the Sf9 insect cells. Examination of synthesis, we used radiolabeled NTPs (Fig. 3B). When POLRMT the purified protein showed that the DNA primase activity was added in isolation we observed only RNA fragments of coincided perfectly with the peak of POLRMT protein during 25–75 nt (Fig.