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Response of a DNA-binding protein to radiation-induced oxidative stress
Françoise Culard1, Alain Gervais, Guillaume de Vuyst
1Centre de biophysique moléculaire, CNRS, rue Charles-Sadron, F-45071 Orléans Cedex 2, France. culard@cnrs-orleans.fr
Abstract:
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.
Insights
Oxidative stress damages the DNA-binding protein MC1, altering its DNA binding and bending capabilities. A proposed model suggests two targets within MC1 are oxidized, leading to functional changes and eventual inactivation.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The DNA-binding protein MC1 from Methanosarcina sp. CHTI55 binds DNA non-specifically but shows higher affinity for certain structures.
- MC1 exhibits strong DNA bending at its binding site.
- MC1 is susceptible to oxidative stress, particularly from hydroxyl radicals generated by gamma-ray irradiation.
Purpose of the Study:
- To investigate the effects of oxidative stress on the DNA-binding protein MC1.
- To elucidate the mechanism of MC1 inactivation under gamma-ray irradiation.
- To propose a model explaining the observed changes in MC1 function and structure.
Main Methods:
- Gamma-ray irradiation of MC1 protein, both free and complexed with DNA.
- Analysis of DNA-protein complex formation and dissociation using titration experiments.
- Characterization of changes in DNA binding affinity, sequence recognition, and DNA bending upon irradiation.
Main Results:
- Irradiation of MC1 leads to the disappearance of the regular DNA-protein complex and the appearance of a new complex.
- In the new complex, MC1 loses preferential sequence and DNA circle recognition and exhibits weaker DNA bending.
- High doses of irradiation result in the complete disappearance of the new complex and total inactivation of the protein.
Conclusions:
- A model involving the oxidation of two targets, R(1) and R(2), in MC1 explains the observed functional modifications and inactivation.
- Oxidation of R(2) has no impact on binding, while R(1) oxidation causes loss of recognition and reduced bending.
- DNA binding protects MC1's binding site from oxidation, and tryptophan and methionine residues are likely involved in the inactivation process.