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Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication.
Proc Natl Acad Sci U S A. 1982 Dec; 79(23):7082-6.PN

Abstract

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.

Authors

No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

6185945

Citation

Selzer, G, and J I. Tomizawa. "Specific Cleavage of the p15A Primer Precursor By Ribonuclease H at the Origin of DNA Replication." Proceedings of the National Academy of Sciences of the United States of America, vol. 79, no. 23, 1982, pp. 7082-6.
Selzer G, Tomizawa JI. Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication. Proc Natl Acad Sci USA. 1982;79(23):7082-6.
Selzer, G., & Tomizawa, J. I. (1982). Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication. Proceedings of the National Academy of Sciences of the United States of America, 79(23), 7082-6.
Selzer G, Tomizawa JI. Specific Cleavage of the p15A Primer Precursor By Ribonuclease H at the Origin of DNA Replication. Proc Natl Acad Sci USA. 1982;79(23):7082-6. PubMed PMID: 6185945.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication. AU - Selzer,G, AU - Tomizawa,J I, PY - 1982/12/1/pubmed PY - 1982/12/1/medline PY - 1982/12/1/entrez SP - 7082 EP - 6 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc. Natl. Acad. Sci. U.S.A. VL - 79 IS - 23 N2 - 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. SN - 0027-8424 UR - https://www.unboundmedicine.com/medline/citation/6185945/Specific_cleavage_of_the_p15A_primer_precursor_by_ribonuclease_H_at_the_origin_of_DNA_replication_ L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=6185945 DB - PRIME DP - Unbound Medicine ER -