Specific Cleavage of the p15A Primer Precursor by Ribonuclease H at the Origin of DNA Replication

We report studies on the mechanism of initiation of DNA replication by p15A, a small plasmid whose origin of replication is known to function much as does that of ColE1. Previous work has shown that an RNA primer for DNA synthesis is generated by the action of RNase H (EC 3.1.26.4) on a precursor transcript. The precursor initiates well upstream of the origin of replication and somehow forms a hybrid with its template during transcription. Here we show that when RNase H cleaves the hybrid at 0 degrees C, an additional cleavage product besides the primer can be identified. Using two-dimensional RNA sequencing techniques, we have established the sequence of this product to within a few nucleotides of each end. The position of the 5′end indicates that the nuclease introduces a nick or very small gap in the precursor at the origin. This suggests that some sequence or structure directs the enzyme to the origin. The position of its 3′end indicates that the precursor terminates at or near a series of six dAs in the template strand about 190 nucleotides from the origin of replication. The data indicate that hybrid formation may be necessary for termination of the precursor at this downstream site.