Primers for vertebrate mitochondrial leading-strand DNA replication are products of transcription synthesized by mitochondrial RNA polymerase. The precursor primer RNA exists as a persistent RNA-DNA hybrid, known as an R-loop, formed during transcription through the replication origin (Xu, B., and Clayton, D. A. (1996) EMBO J. 15, 3135-3143). In an effort to examine the precise structure of this primer RNA intermediate, we have used two methods to reconstitute model R-loops containing the mouse

more »

... mitochondrial DNA origin sequence. First, we demonstrate that bacteriophage SP6 RNA polymerase can efficiently catalyze the formation of an R-loop at the mouse mtDNA origin sequence. Second, the R-loop can be assembled by annealing presynthesized RNA and supercoiled DNA template in the presence of formamide. R-loop formation by either method is dependent on specific template sequences. The reconstituted R-loop is exceptionally stable and exhibits an unexpected structure. Structural studies indicate that the RNA strand is organized within the RNA-DNA basepaired region, suggesting that the heteroduplex interaction occurs through a specific conformation. We propose that the organized structure of the R-loop is critical for primer RNA function in vivo with important implications for the RNA processing and DNA replication machinery. The mammalian mitochondrial genome (mtDNA) is a small, closed circular molecule of ϳ16,000 base pairs (1, 2). Each strand of this compact supercoiled genome is transcribed from a single major promoter, the heavy-strand promoter and the light-strand promoter (LSP). 1 Analyses of newly replicated DNA strands from the mtDNA control region (called the "Dloop region"), which houses both promoters and the leadingstrand replication origin (O H ), have revealed an intimate relationship between transcription and the initiation of DNA synthesis. All of the major nascent DNA strands were found to initiate within ϳ200 nucleotides downstream of the LSP in human and mouse mitochondria (3, 4). For both systems, two species of LSP transcripts were observed: transcripts that extended beyond the replication start sites and transcripts with 3Ј-termini mapping precisely at or closely to the start sites of nascent DNA strands. In the mouse system, a third type of LSP transcript was demonstrated: those that were covalently attached at the 5Ј-termini of nascent DNA strands (4), confirming earlier observations of RNA-linked DNAs from the O H region (5). These findings supported a model for transcriptional activation of leading-strand DNA replication. Recent results from in vitro transcription experiments using purified mitochondrial RNA polymerase (RNAP) activities have suggested that origin activation occurs through the formation of a persistent RNA-DNA hybrid at a specific region encompassing the sites of DNA replication (6). This persistent RNA-DNA hybrid, also termed an "R-loop," has been postulated to represent the precursor form of the primer RNA that, upon endonucleolytic processing, leads to mature primers that are properly positioned on the template for DNA synthesis. To understand the physical basis for the stable RNA-DNA interaction and to facilitate in vitro studies of primer RNA metabolism, we have reconstituted a model R-loop with the mouse mtDNA O H sequence. In this report, we describe two methods of generating a stable triple-stranded complex of RNA and plasmid DNA. We first show that bacteriophage SP6 RNAP can substitute for mitochondrial RNAP in forming an R-loop at a discrete region that encompasses all of the known DNA synthesis start sites at the mouse mtDNA O H . We then demonstrate that an R-loop can be assembled using a defined length RNA synthesized in vitro and annealing it onto a supercoiled plasmid template in the presence of a mild denaturant. The reconstituted R-loop is resistant to branch migration and can tolerate temperatures of up to 95°C. Due to this stability, the R-loop can be purified to homogeneity by conventional liquid chromatography. Nuclease probing assays indicate that the RNA strand in the R-loop is precisely positioned on the DNA template and is folded in a configuration that is similar to its free form in solution. We propose that the RNA conformation facilitates the RNA-DNA interaction, which stabilizes the precursor primer RNA on the DNA template. EXPERIMENTAL PROCEDURES Nucleic Acids-pMR718B contains an RsaI fragment of mouse mtDNA as described previously (7). pMR718X.2 was constructed by subcloning the mtDNA fragment of pMR178B into the BamHI-PvuII sites of pSP65 such that SP6 transcription generates RNA of the reverse orientation relative to that of pMR718B. Construction of pMR718B derivatives with mutations or deletions of CSB II or CSB III was described previously (8). Plasmids were grown in DH5␣ and purified by two consecutive CsCl/ethidium bromide gradients to obtain the superhelical form. Buoyant density gradient-purified DNA was passed through Sepharose CL-4B (Pharmacia Biotech Inc.) and quantitated by UV absorption. A 338-nucleotide run-off RNA, corresponding to the mtDNA light-strand transcript, was synthesized from pMR718B that had been digested with NsiI endonuclease (New England Biolabs Inc.) using 40 -80 units of SP6 RNA polymerase in a 50 -100-l reaction volume containing 0.5 mM each of four ribonucleoside triphosphates, 40 mM Tris (pH 7.9), 10 mM MgCl 2 , 1 mM spermidine, and 1 mM dithio-