High-LET irradiation of a DNA-binding protein: protein-protein and DNA-protein crosslinks.

The chromosomal protein MC1 is a monomeric protein of 93 amino acids that is able to bind any DNA but has a slight preferential affinity for some sequences and structures, like cruciform and minicircles. The protein has been irradiated with 36Ar18+ ions of 95 MeV/nucleon. The LET of these particles in water is close to 270 keV/microm. We tested the activity of the protein by measuring its ability to form complexes with DNA. We tested the integrity of the protein by measuring the molecular weight of the species formed. Compared with gamma radiation, we observed for the same dose a less efficient inactivation of the protein, a greater protection of the protein by the bound DNA, a lower induction of chain breakage, and a greater production of protein-protein and DNA-protein crosslinks. The results are discussed in terms of the quantitative and the qualitative differences between the two types of radiation: The global radical yield is slightly higher with gamma rays, whereas the density of radicals produced along the particle track is considerably higher with argon ions.

Radiation abolishes inducer binding to lactose repressor.

The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that gamma irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-beta-D-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects.

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.

Direct effect in DNA radiolysis. Boron neutron capture enhancement of radiolysis in a medical fast-neutron beam.

SècheThis paper is devoted to the study of the molecular basis of the boron neutron capture enhancement of fast-neutron radiotherapy. Plasmid DNA was irradiated with a medical fast-neutron beam in the presence of either (10)B or (11)B. The number of induced SSBs and DSBs was much higher in samples containing (10)B compared to (11)B. The additional breaks are attributed to the nuclear reaction (10)B(n, alpha)(7)Li induced by the capture by (10)B of thermal neutrons produced in the medium by scattering and slowing down of neutrons. Irradiation in the presence of DMSO (OH radical scavenger) allows the number of nonscavengeable breaks to be determined. The ratio DSB/SSB is within the range of those observed with heavy ions, in good agreement with the hypothesis that the additional breaks are due to alpha particles and recoil lithium nuclei. The simulation of the energy deposition along the paths of the alpha and (7)Li particles allows the calculation of core and penumbra track volumes. Further, the number of plasmids encountered by the core and the penumbra was evaluated. Their number was compared to the nonscavengeable additional breaks. Since the two sets of values are of the same order of magnitude, we conclude that the nonscavengeable additional SSBs and DSBs could be due to direct effects.

Radiolysis of lac repressor by gamma-rays and heavy ions: a two-hit model for protein inactivation.

Upon gamma-ray or argon ion irradiation of the lac repressor protein, its peptide chain is cleaved and the protein loses its lac operator-binding activity, as shown respectively by polyacrylamide gel electrophoresis and retardation gel electrophoresis. We developed phenomenological models that satisfactorily account for the experimental results: the peptide chain cleavage model considers that the average number of chain breaks per protomer is proportional to the irradiation dose and that the distribution of the number of breaks per protomer obeys Poisson's law. The repressor inactivation model takes into account the quaternary structure (a dimer of dimer) and the organization of the repressor in domains (two DNA binding sites, one per dimer). A protomer is inactivated by at least two different radiation-induced damages. A dimer is inactivated when at least one of the two protomers is inactivated. A tetramer is inactivated when both dimers are inactivated. From the combination of both models, we can deduce that chain cleavage cannot account for the protein inactivation, which should mainly result from oxidation of amino acid side chains. Indeed, particularly oxidizable and accessible amino acids (Tyr, His) are involved in the DNA binding process.