Alanine screening mutagenesis establishes the critical inactivating damage of irradiated E. coli lactose repressor.

The function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor.

Comparing native and irradiated E. coli lactose repressor-operator complex by molecular dynamics simulation.

The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that gamma-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604-612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions.

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor.

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.

Modification of DNA radiolysis by DNA-binding proteins: structural aspects.

Formation of specific complexes between proteins and their cognate DNA modulates the yields and the location of radiation damage on both partners of the complex. The radiolysis of DNA-protein complexes is studied for: (1) the Escherichia coli lactose operator-repressor complex, (2) the complex between DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus lactis. Experimental patterns of DNA damages are presented and compared to predicted damage distribution obtained using an improved version of the stochastic model RADACK. The same method is used for predicting the location of damages on the proteins. At doses lower than a threshold that depends on the system, proteins protect their specific binding site on DNA while at high doses, the studied complexes are disrupted mainly through protein damage. The loss of binding ability is the functional consequence of the amino-acids modification by OH* radicals. Many of the most probably damaged amino acids are essential for the DNA-protein interaction and within a complex are protected by DNA.

Radiation damage to DNA-protein specific complexes: estrogen response element-estrogen receptor complex.

The exposure of a DNA-protein regulatory complex to ionising radiation induces damage to both partner biomolecules and thus can affect its functioning. Our study focuses on a complex formed by the estrogen response element (ERE) DNA and the recombinant human estrogen receptor alpha (ER), which mediates the signalling of female sex hormones, estrogens. The method of native polyacrylamide retardation gel electrophoresis is used to study the stability of the complex under irradiation by low LET radiation ((60)Co gamma rays) and the ability of the separately irradiated partners to form complexes. The relative probabilities of ERE DNA strand breakage and base damages as well as the probabilities of damages to the ER binding domain are calculated using the Monte Carlo method-based model RADACK.

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.

Radiation affects binding of Fpg repair protein to an abasic site containing DNA.

During the base excision repair of certain DNA lesions, the formamidopyrimidine-DNA glycosylase (Fpg) binds specifically to the DNA region containing an abasic (AP) site. Is this step affected by exposure to ionizing radiation? To answer this question, we studied a complex between a DNA duplex containing an analogue of an abasic site (the 1,3-propanediol site, Pr) and a mutated Lactococcus lactis Fpg (P1G-LlFpg) lacking strand cleavage activity. Upon irradiation of the complex, the ratio of bound/free partners decreased. When the partners were irradiated separately, the irradiated DNA still bound the unirradiated protein, whereas irradiated Fpg no longer bound unirradiated DNA. Thus irradiation hinders Fpg-DNA binding because of the damage to the protein. Using our radiolytic attack simulation procedure RADACK (Begusova et al., J. Biomol. Struct. Dyn. 19, 141-157, 2001), we reveal the potential hot spots for damage in the irradiated protein. Most of them are essential for the interaction of Fpg with DNA, which explains the radiation-induced loss of binding ability of Fpg. The doses necessary to destroy the complex are higher than those inactivating Fpg irradiated separately. As confirmed by our calculations, this can be explained by the partial protection of the protein by the bound DNA.

In vitro studies with methylproamine: a potent new radioprotector.

New analogues of the minor groove binding ligand Hoechst 33342 have been investigated in an attempt to improve radioprotective activity. The synthesis, DNA binding, and in vitro radioprotective properties of methylproamine, the most potent derivative, are reported. Experiments with V79 cells have shown that methylproamine is approximately 100-fold more potent than the classical aminothiol radioprotector WR1065. The crystal structures of methylproamine and proamine complexes with the dodecamer d(CGCGAATTCGCG)(2) confirm that the new analogues also are minor groove binders. It is proposed that the DNA-bound methylproamine ligand acts as a reducing agent by an electron transfer mechanism, repairing transient radiation-induced oxidizing species on DNA.

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.