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. 1977 Jun;4(6):1803–1813. doi: 10.1093/nar/4.6.1803

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases.

G Ruben, P Spielman, C D Tu, E Jay, B Siegel, R Wu
PMCID: PMC342523  PMID: 197493

Abstract

We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.

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Selected References

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

  1. Greene P. H., Poonian M. S., Nussbaum A. L., Tobias L., Garfin D. E., Boyer H. W., Goodman H. M. Restriction and modification of a self-complementary octanucleotide containing the EcoRI substrate. J Mol Biol. 1975 Dec 5;99(2):237–261. doi: 10.1016/s0022-2836(75)80143-4. [DOI] [PubMed] [Google Scholar]
  2. Gromkova R., Goodgal S. H. Action of haemophilus endodeoxyribonuclease on biologically active deoxyribonucleic acid. J Bacteriol. 1972 Mar;109(3):987–992. doi: 10.1128/jb.109.3.987-992.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hedgpeth J., Goodman H. M., Boyer H. W. DNA nucleotide sequence restricted by the RI endonuclease. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3448–3452. doi: 10.1073/pnas.69.11.3448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Mertz J. E., Davis R. W. Cleavage of DNA by R 1 restriction endonuclease generates cohesive ends. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3370–3374. doi: 10.1073/pnas.69.11.3370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Modrich P., Zabel D. EcoRI endonuclease. Physical and catalytic properties of the homogenous enzyme. J Biol Chem. 1976 Oct 10;251(19):5866–5874. [PubMed] [Google Scholar]
  6. Mulder C., Delius H. Specificity of the break produced by restricting endonuclease R 1 in Simian virus 40 DNA, as revealed by partial denaturation mapping. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3215–3219. doi: 10.1073/pnas.69.11.3215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  8. Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]
  9. Wu R., Ruben G., Siegel B., Jay E., Spielman P., Tu C. P. Synchronous digestion of SV40 DNA by exonuclease III. Biochemistry. 1976 Feb 24;15(4):734–740. doi: 10.1021/bi00649a003. [DOI] [PubMed] [Google Scholar]

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