Response of a DNA-binding protein to radiation-induced oxidative stress.
The DNA-binding protein MC1 is a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55. It binds any DNA, and exhibits an enhanced affinity for some short sequences and structures (circles, cruciform DNA). Moreover, the protein bends DNA strongly at the binding site. MC1 was submitted to oxidative stress through gamma-ray irradiation. In our experimental conditions, damage is essentially due to hydroxyl radicals issued from water radiolysis. Upon irradiation, the regular complex between MC1 and DNA disappears, while a new complex appears. In the new complex, the protein loses its ability to recognise preferential sequences and DNA circles, and bends DNA less strongly than in the regular one. The new complex disappears and the protein becomes totally inactivated by high doses.A model has been proposed to explain these experimental results. Two targets, R(1) and R(2), are concomitantly destroyed in the protein, with different kinetics. R(2) oxidation has no effect on the regular binding, whereas R(1) oxidation modifies the functioning of MC1: loss of preferential site and structure recognition, weaker bending. The destruction of both R(1) and R(2) targets leads to a total inactivation of the protein. This model accounts for the data obtained by titrations of DNA with irradiated proteins. When the protein is irradiated in the complex with DNA, bound DNA protects its binding site on the protein very efficiently. The highly oxidisable tryptophan and methionine could be the amino acid residues implicated in the inactivation process.
Culard
Françoise
F
Centre de biophysique moléculaire, CNRS, rue Charles-Sadron, F-45071 Orléans Cedex 2, France. culard@cnrs-orleans.fr
Gervais
Alain
A
de Vuyst
Guillaume
G
Spotheim-Maurizot
Mélanie
M
Charlier
Michel
M
D019843:Archaeal Proteins / Q000378:metabolism* / Q000528:radiation effects; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000528:radiation effects; D007700:Kinetics; D017020:Methanosarcina / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D008954:Models, Biological; D018384:Oxidative Stress / Q000528:radiation effects*; D012261:Ribonucleoproteins / Q000378:metabolism* / Q000528:radiation effects
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004270:DNA, Circular; D004268:DNA-Binding Proteins; C051211:HMb protein, Methanosarcina; D012261:Ribonucleoproteins