Ultraviolet Footprinting'' Accurately Maps Sequence-Specific Contacts and DNA Kinking in the EcoRI Endonuclease-DNA Complex

The ``UV footprinting'' technique has been used to detect contacts between EcoRI endonuclease and its recognition sequence at single nucleotide resolution. Comparison of the UV-footprinting results to the published crystal structure of the EcoRI endonuclease-DNA complex allows us to determ...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1988-09, Vol.85 (17), p.6247-6251
Main Authors: Becker, Michael M., Lesser, David, Kurpiewski, Michael, Baranger, Anne, Jen-Jacobson, Linda
Format: Article
Language:English
Subjects:
Base Sequence
Biological and medical sciences
Chemical bases
Crystal structure
Deoxyribonuclease EcoRI
Dimers
DNA
DNA - metabolism
DNA Restriction Enzymes - metabolism
Fundamental and applied biological sciences. Psychology
Gels
Interactions. Associations
Intermolecular phenomena
Irradiation
Molecular biophysics
Nucleic Acid Conformation
Oligonucleotides
Phosphates
Photochemistry
Protein Binding
Pyrimidines
Radiocarbon
Spectrophotometry, Ultraviolet - methods
X-Ray Diffraction
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Summary:The ``UV footprinting'' technique has been used to detect contacts between EcoRI endonuclease and its recognition sequence at single nucleotide resolution. Comparison of the UV-footprinting results to the published crystal structure of the EcoRI endonuclease-DNA complex allows us to determine how UV light detects protein-DNA contacts. We find that kinking of the DNA helix in the complex greatly enhances the UV photoreactivity of DNA at the site of the kink. In contrast to kinking, contacts between the endonuclease and the DNA bases inhibit the UV photoreactivity of DNA. Similar analysis of a proteolytically modified endonuclease that exhibits the same sequence specificity as wild-type enzyme but that does not cleave DNA supports these conclusions. Furthermore, detection of enhanced photoreactivity at the same kink in the modified enzyme-DNA complex allows us to conclude that the loss of cleavage activity by the modified endonuclease is not due to its failure to kink DNA.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.85.17.6247