| First Author | Lee DY | Year | 1996 |
| Journal | J Biol Chem | Volume | 271 |
| Issue | 39 | Pages | 24262-9 |
| PubMed ID | 8798672 | Mgi Jnum | J:35628 |
| Mgi Id | MGI:83075 | Doi | 10.1074/jbc.271.39.24262 |
| Citation | Lee DY, et al. (1996) Properties of a primer RNA-DNA hybrid at the mouse mitochondrial DNA leading-strand origin of replication. J Biol Chem 271(39):24262-9 |
| abstractText | 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 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 base-paired 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. |
