RNA-DNA HYBRID FORMATION AT THE HUMAN MITOCHONDRIAL HEAVY-STRAND ORIGIN CEASES AT REPLICATION START SITES - AN IMPLICATION FOR RNA-DNA HYBRIDS SERVING AS PRIMERS

Critical elements of a mammalian mitochondrial DNA heavy-strand replic ation origin include a promoter and three downstream conserved sequenc e blocks (CSBIII, CSBII and CSBI), We found recently that a stable and persistent RNA-DNA hybrid forms during in vitro transcription at Sacc haromyces cerevisiae mitochondrial origins; hybrid formation was depen dent on the conserved CSBII element, We report here that during in vit ro transcription with human mitochondrial RNA polymerase, stable and p ersistent RNA-DNA hybrid formation is also evident at the human mitoch ondrial heavy-strand origin, As predicted, hybrid formation was depend ent on the GC-rich CSBII element. The human RNA-DNA hybrids terminate within or downstream of CSBI at locations implicated in initiation of mitochondrial DNA replication, Interestingly, efficient hybrid formati on in the human system is influenced by sequence 5' to the RNA-DNA hyb rid, including the CSBIII element, These results suggest that the RNA- DNA hybrids formed during transcription across the mitochondrial DNA h eavy-strand origin provide RNA primers for initiation of mitochondrial DNA replication.