Initiation of Mitochondrial DNA Replication by Transcription and R-loop Processing

Daniel Y Lee,David A Clayton

doi:10.1074/jbc.273.46.30614

Abstract

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.

Highlights

Transcription of mtDNA is directed by cis-acting elements located at the 5Ј boundary of the regulatory sequence known as the D-loop, a region which houses the leading-strand origin of replication, OH [1]
The results of our studies suggest that the initiation of DNA replication at the mammalian mtDNA OH involves transcription-coupled synthesis of a structurally complex pre-primer RNA that remains stably hybridized to the DNA template
Since we have found that RNase MRP cleaves at virtually all of the known priming sites, the action of this single enzyme can account for the heterogeneous primer RNA species observed in vivo

Summary

Introduction

Transcription of mtDNA is directed by cis-acting elements located at the 5Ј boundary of the regulatory sequence known as the D-loop, a region which houses the leading-strand origin of replication, OH [1]. Consistent with previous results, the observed R-loop processing activity was abolished (Fig. 2A, lane 7), demonstrating a requirement for RNA in the R-loop cleavage reaction. Since we have previously shown that oligo LRI had no inhibitory effect on the related ribonucleoprotein endonuclease RNase P, these data suggest that the observed R-loop processing reaction is catalyzed by RNase MRP [8].

Results

Conclusion