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Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies
Departments of Biological Sciences, The University of Calgary Calgary, Alberta, Canada.
Medical Biochemistry, The University of Calgary Calgary, Alberta, Canada.
Linköping University, Department of Medical and Health Sciences, Division of Radiological Sciences. Linköping University, Faculty of Health Sciences. Linköping University, Center for Medical Image Science and Visualization (CMIV). Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Radiation Physics. Departments of Biological Sciences, The University of Calgary Calgary, Alberta, Canada.ORCID iD: 0000-0001-8661-2232
Departments of Biological Sciences, The University of Calgary Calgary, Alberta, Canada.
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1990 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 18, no 6, 1489-1498 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.

Place, publisher, year, edition, pages
Oxford University Press, 1990. Vol. 18, no 6, 1489-1498 p.
Keyword [en]
*Dna, Deoxyribonuclease EcoRI/*metabolism, Electrophoresis, Polyacrylamide Gel, Magnesium/pharmacology, Magnetic Resonance Spectroscopy/methods, *Nucleic Acid Conformation, Nucleic Acid Denaturation, *Oligodeoxyribonucleotides/chemical synthesis, Spectrophotometry, Ultraviolet/methods, Substrate Specificity, Thermodynamics
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:liu:diva-114166DOI: 10.1093/nar/18.6.1489PubMedID: 2326190OAI: oai:DiVA.org:liu-114166DiVA: diva2:790768
Note

0305-1048 (Print) Journal Article Research Support, Non-U.S. Gov't

Available from: 2015-02-25 Created: 2015-02-11 Last updated: 2017-12-04Bibliographically approved

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Lundberg, Peter

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Division of Radiological SciencesFaculty of Health SciencesCenter for Medical Image Science and Visualization (CMIV)Department of Radiation Physics
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