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(PP028) Radiolysis of DNA-binding proteins : experiment versus calculation.
Gillard, Nathalie 1, Begusova, Marie2, Castaing, Bertrand1, Culard, Francoise1, Spotheim-Maurizot, Melanie *,1, 1 Centre de Biophysique Moleculaire, Orleans, France, France2 Department of Radiation Dosimetry, Prague 8, Czech Republic, Czech Republic
ABSTRACT- Binding of proteins to their cognate DNA sequences is a key step in the regulation of gene expression, DNA structuring and DNA repair. These processes may be disturbed if radiation destroys the DNA-protein complexes by damaging one or both partners. We have studied the effect of irradiation on three systems : 1) the E. coli lactose operator-repressor complex, 2) the complex between MC1, a DNA structuring protein of Methanosarcina, and its cognate DNA sequence and 3) the complex between a DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus Lactis. At low doses the proteins protect their specific binding site on DNA from damage. For the lactose repressor and for the Fpg protein, the experimentally revealed protected regions are the same as those predicted by calculation with our up-dated RADACK model for structures of the complexes from PDB Databank ((Begusova et al. Radiat. Phys.Chem., 2005, 72, 265-270). At high doses, the three studied complexes are disrupted mainly due to protein damage. The irradiation of the free proteins induces the loss of their ability to bind DNA at even lower doses than those necessary to the disruption of the irradiated complexes. This difference of dose is due to the protection, in the complex, of the proteins by the bound DNA. The loss of binding ability is the functional consequence of the modification of the amino-acids by the OH radicals produced by water radiolysis. Using RADACK we have identified the most probable sites of damage on the free proteins. Many of the most probably damaged amino-acids are essential for the DNA-protein interaction as observed on the NMR- or crystallography-based structures of the complexes. Calculations show also that as long as the complexes still exist these essential amino-acids are protected by DNA.
Key words: DNA binding proteins, RADACK model, structural data, damaged amino-acids
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