Properties of a primer RNA-DNA hybrid at the mouse mitochondrial DNA leading-strand origin of replication.
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.
Lee
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Department of Developmental Biology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5427, USA.
Clayton
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D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D006860:Hydrogen Bonding; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D016914:Ribonuclease H / Q000378:metabolism; D014158:Transcription, Genetic
D004272:DNA, Mitochondrial; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H