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. 1982 Dec;79(23):7082–7086. doi: 10.1073/pnas.79.23.7082

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

G Selzer, J I Tomizawa
PMCID: PMC347282  PMID: 6185945

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berkower I., Leis J., Hurwitz J. Isolation and characterization of an endonuclease from Escherichia coli specific for ribonucleic acid in ribonucleic acid-deoxyribonucleic acid hybrid structures. J Biol Chem. 1973 Sep 10;248(17):5914–5921. [PubMed] [Google Scholar]
  2. Bird R. E. Homology between Escherichia coli plasmids ColE1 and p15A. J Bacteriol. 1981 Mar;145(3):1305–1309. doi: 10.1128/jb.145.3.1305-1309.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  4. Cozzarelli N. R., Kelly R. B., Kornberg A. A minute circular DNA from Escherichia coli 15. Proc Natl Acad Sci U S A. 1968 Jul;60(3):992–999. doi: 10.1073/pnas.60.3.992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dirksen M. L., Crouch R. J. Selective inhibition of RNase H by dextran. J Biol Chem. 1981 Nov 25;256(22):11569–11573. [PubMed] [Google Scholar]
  6. Donis-Keller H. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 1979 Sep 11;7(1):179–192. doi: 10.1093/nar/7.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Itoh T., Tomizawa J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A. 1980 May;77(5):2450–2454. doi: 10.1073/pnas.77.5.2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Itoh T., Tomizawa J. Initiation of replication of plasmid ColE1 DNA by RNA polymerase, ribonuclease H, and DNA polymerase I. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):409–417. doi: 10.1101/sqb.1979.043.01.047. [DOI] [PubMed] [Google Scholar]
  9. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  10. Robertson H. D., Dunn J. J. Ribonucleic acid processing activity of Escherichia coli ribonuclease III. J Biol Chem. 1975 Apr 25;250(8):3050–3056. [PubMed] [Google Scholar]
  11. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  12. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  13. Staudenbauer W. L. Structure and replication of the colicin E1 plasmid. Curr Top Microbiol Immunol. 1978;83:93–156. doi: 10.1007/978-3-642-67087-9_3. [DOI] [PubMed] [Google Scholar]
  14. Tomizawa J., Itoh T. Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6096–6100. doi: 10.1073/pnas.78.10.6096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tomizawa J., Itoh T., Selzer G., Som T. Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1421–1425. doi: 10.1073/pnas.78.3.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Zimmerman S. B., Pheiffer B. H. A RNA.DNA hybrid that can adopt two conformations: an x-ray diffraction study of poly(rA).poly(dT) in concentrated solution or in fibers. Proc Natl Acad Sci U S A. 1981 Jan;78(1):78–82. doi: 10.1073/pnas.78.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]

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