Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication.

Abstract

The mechanism of initiation of DNA replication by [the Escherichia coli] plasmid p15A, a small plasmid whose origin of replication is known to function much as does that of ColE1, was studied. An RNA primer for DNA synthesis is generated by the action of RNase H (EC 3.1.26.4) on a precursor transcript. The percursor initiated well upstream of the origin of replication and somehow forms a hybrid with its template during transcription. When RNase H cleaves the hybrid at 0.degree. C, an additional cleavage product besides the primer can be identified. Using 2-dimensional RNA sequencing techniques, the sequence of this product was established 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. The position of its 3'' end indicates that the precursor terminates at or near a series of 6 dA (deoxyadenylic acid residues) in the template strand about 190 nucleotides from the origin of replication. Hybrid formation may be necessary for termination of the precursor at this downstream site.