A PERSISTENT RNA-DNA HYBRID IS FORMED DURING TRANSCRIPTION AT A PHYLOGENETICALLY CONSERVED MITOCHONDRIAL-DNA SEQUENCE

Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans. These in clude a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA. During in vitro transcription at ye ast mitochondrial replication origins, there is stable and persistent RNA-DNA hybrid formation that begins at the 5' end of the rG region. T he short rG-dC sequence is the necessary and sufficient nucleic acid e lement for establishing stable hybrids, and the presence of rGs within the RNA strand of the RNA-DNA hybrid is required. The efficiency of h ybrid formation depends on the length of RNA synthesized 5' to CSBII a nd the type of RNA polymerase employed. Once made, the RNA strand of a n RNA-DNA hybrid can serve as an effective primer for mitochondrial DN A polymerase. These results reveal a new mechanism for persistent RNA- DNA hybrid formation and suggest a step in priming mitochondrial DNA r eplication that requires both mitochondrial RNA polymerase and an rG-d C sequence-specific event to form an extensive RNA-DNA hybrid.