Initiation of mitochondrial DNA replication by transcription and R-loop processing.
The mitochondrial genome of eukaryotic cells is maintained by a mechanism distinct from that employed in the nucleus. Mitochondrial DNA replication at the leading-strand origin is coupled to transcription through the formation of an RNA-DNA hybrid known as an R-loop. In vivo and in vitro evidence has implicated an RNA processing enzyme, RNase MRP, in primer maturation. In our investigation of mammalian RNase MRP, we have analyzed its specific endoribonuclease activity on model R-loops. We demonstrate here that human RNase MRP cleaves this distinctly configured substrate at virtually all of the major DNA replication sites previously mapped in vivo. We further show that the processed RNA products remain stably base-paired to the template DNA strand and are functional for initiating DNA synthesis on a closed circular plasmid. Thus, in vitro initiation of leading-strand mtDNA synthesis requires only the actions of RNA polymerase and RNase MRP for the generation of replication primers.
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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D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D004722:Endoribonucleases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D014158:Transcription, Genetic
D004270:DNA, Circular; D004272:DNA, Mitochondrial; D012261:Ribonucleoproteins; D012313:RNA; D004722:Endoribonucleases; C058330:mitochondrial RNA-processing endoribonuclease