Radiolytic footprinting. Beta rays, gamma photons, and fast neutrons probe DNA-protein interactions.
Ionizing radiations induce numerous damages in DNA, especially strand breaks. The hydroxyl radical OH., produced by the radiolysis of water, is mainly responsible for this effect. The fact that strand breakage occurs at all nucleotides and that bound proteins may locally radioprotect DNA at the binding site lead us to develop a radiolytic footprinting method to study DNA-protein interactions. Three different radiations were used: beta rays, gamma photons, and fast neutrons. In order to validate this technique, three well-known interaction systems were tested: the lac repressor-lac operator of Escherichia coli, the cyclic AMP receptor protein (CRP) of E. coli and its specific site in the lac regulation region, and the core nucleosome. Radiolytic footprinting gives results similar to those obtained by more classical probes: DNase I, complexes of orthophenanthroline (OP) and copper, complexes of ethylenediaminetetraacetate ion (EDTA) and iron, and UV light. For the same system (lac repressor), irradiation with either gamma photons or fast neutrons gives identical results.
Franchet-Beuzit
J
J
Centre de Biophysique Moléculaire, CNRS, Orléans, France.
Spotheim-Maurizot
M
M
Sabattier
R
R
Blazy-Baudras
B
B
Charlier
M
M
D000818:Animals; D001483:Base Sequence; D001610:Beta Particles; D002352:Carrier Proteins / Q000378:metabolism* / Q000528:radiation effects; D002645:Chickens; D002373:Cyclic AMP Receptor Protein; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005609:Free Radicals; D005720:Gamma Rays; D006878:Hydroxides; D017665:Hydroxyl Radical; D007763:Lac Operon / Q000528:radiation effects; D008969:Molecular Sequence Data; D009502:Neutrons; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000378:metabolism; D010957:Plasmids / Q000528:radiation effects; D011953:Receptors, Cyclic AMP / Q000378:metabolism* / Q000528:radiation effects; D012097:Repressor Proteins / Q000378:metabolism* / Q000528:radiation effects; D015183:Restriction Mapping; D014466:Ultraviolet Rays
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D005609:Free Radicals; D006878:Hydroxides; D009707:Nucleosomes; D011953:Receptors, Cyclic AMP; D012097:Repressor Proteins; D017665:Hydroxyl Radical; D004247:DNA