INITIATION OF MITOCHONDRIAL-DNA REPLICATION BY TRANSCRIPTION AND R-LOOP PROCESSING

The mitochondrial genome of eukaryotic cells is maintained by a mechan ism distinct from that employed in the nucleus. Mitochondrial DNA repl ication at the leading-strand origin is coupled to transcription throu gh the formation of an RNA-RNA 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 conf igured substrate at virtually all of the major DNA replication sites p reviously mapped in vivo. We further show that the processed RNA produ cts remain stably base-paired to the template DNA strand and are funct ional for initiating DNA synthesis on a closed circular plasmid. Thus, in vitro initiation of leading-strand mtDNA synthesis requires only t he actions of RNA polymerase and RNase MRP for the generation of repli cation primers.