Radiosensitivity of DNA in a specific protein-DNA complex: the lac repressor-lac operator complex.
OBJECTIVE
To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor-lac operator system using a simulation procedure. To compare calculated and experimental results. To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor.


METHODS
Two structures of the complex were extracted from the PDB databank: crystallography- and NMR-based structures. Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4' and H5') to O&Hdot; and of the FSB probabilities, along: (1) DNA in the complex; (2) DNA in the complex depleted of the repressor; and (3) a linear DNA having the same sequence. An 80bp fragment bearing the operator was irradiated alone or in presence of the repressor. The relative probabilities of FSB at each nucleotide were determined using sequencing gel electrophoresis.


RESULTS
Calculations predict modulation of the accessibility of H4' and H5' atoms and of the probabilities of FSB along the DNA fragments of complexes. This is due to the protein-induced conformational change and to masking by bound protein. The best agreement with the experimental FSB was observed for calculations that use the crystallography-based structure.


CONCLUSIONS
For specific DNA-protein complexes, our calculations can predict the protein radiolytic footprints on DNA. They show the significant contribution of the protein-induced DNA conformational change to DNA protection.
Begusová
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Nuclear Physics Institute, Radiation Protection Department, Na Truhlarce 39, CZ-18086, Praha 8, Czech Republic.
Eon
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Culard
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Charlier
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Spotheim-Maurizot
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D001426:Bacterial Proteins / Q000737:chemistry / Q000528:radiation effects*; D001483:Base Sequence; D004249:DNA Damage; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects; D029968:Escherichia coli Proteins; D017665:Hydroxyl Radical / Q000737:chemistry / Q000528:radiation effects; D007763:Lac Operon / Q000528:radiation effects*; D056968:Lac Repressors; D046911:Macromolecular Substances; D008958:Models, Molecular; D009010:Monte Carlo Method; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000528:radiation effects; D011487:Protein Conformation; D011836:Radiation Tolerance; D012097:Repressor Proteins / Q000737:chemistry / Q000528:radiation effects*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D046911:Macromolecular Substances; D012097:Repressor Proteins; D017665:Hydroxyl Radical