Radiation disrupts protein-DNA complexes through damage to the protein. The lac repressor-operator system.

Eon, S., Culard, F., Sy, D., Charlier, M. and Spotheim-Maurizot, M. Radiation Disrupts Protein-DNA Complexes through Damage to the Protein. The lac Repressor-Operator System. Radiat. Res. 156, 110-117 (2001). Binding of a protein to its cognate DNA sequence is a key step in the regulation of gene expression. If radiation damage interferes with protein-DNA recognition, the entire regulation process may be perturbed. We have studied the effect of gamma rays on a model regulatory system, the E. coli lactose repressor-operator complex. We have observed the disruption of the complex upon irradiation in aerated solution. The complex is completely restored by the addition of nonirradiated repressor, but not by the addition of nonirradiated DNA. Thus radiation disrupts the DNA-protein complex by affecting the binding ability of the protein. This interpretation is supported by the dramatic loss of binding ability of a free irradiated repressor toward nonirradiated DNA. Interestingly, the dose necessary for the disruption of the irradiated complex is higher than that for inducing the complete loss of the binding ability of the free irradiated repressor. This may be due to the protection of key amino acids by the bound DNA. As seen from calculations of the accessibility of amino acids to radiolytic OH(.), the protection is due to both masking and conformational effects.

Radiosensitivity of DNA in a specific protein-DNA complex: the lac repressor-lac operator complex.

PURPOSE: To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor-lac operator system using a simulation procedure. To compare calculated and experimental results. To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor. MATERIALS AND METHODS: Two structures of the complex were extracted from the PDB databank: crystallography- and NMR-based structures. Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4' and H5') to O&Hdot; and of the FSB probabilities, along: (1) DNA in the complex; (2) DNA in the complex depleted of the repressor; and (3) a linear DNA having the same sequence. An 80bp fragment bearing the operator was irradiated alone or in presence of the repressor. The relative probabilities of FSB at each nucleotide were determined using sequencing gel electrophoresis. RESULTS: Calculations predict modulation of the accessibility of H4' and H5' atoms and of the probabilities of FSB along the DNA fragments of complexes. This is due to the protein-induced conformational change and to masking by bound protein. The best agreement with the experimental FSB was observed for calculations that use the crystallography-based structure. CONCLUSIONS: For specific DNA-protein complexes, our calculations can predict the protein radiolytic footprints on DNA. They show the significant contribution of the protein-induced DNA conformational change to DNA protection.

DNA bending induced by the archaebacterial histone-like protein MC1.

The conformational changes induced by the binding of the histone-like protein MC1 to DNA duplexes have been analyzed by dark-field electron microscopy and polyacrylamide gel electrophoresis. Visualisation of the DNA molecules by electron microscopy reveals that the binding of MC1 induces sharp kinks. Linear DNA duplexes (176 bp) which contained a preferential site located at the center were used for quantitative analysis. Measurements of the angle at the center of all duplexes, at a fixed DNA concentration, as a function of the MC1 concentration, were very well fitted by a simple model of an isotropic flexible junction and an equilibrium between the two conformations of DNA with bound or unbound MC1. This model amounts to double-folded Gaussian distributions and yields an equilibrium deflection angle of theta0=116 degrees for the DNA with bound MC1. It allowed measurements of the fraction of DNA with bound MC1 to be taken as a function of MC1 concentrations and yields an equilibrium dissociation constant of Kd=100 nM. It shows that the flexibility of DNA is reduced by the binding of MC1 and the formation of a kink. The equilibrium dissociation constant value was corroborated by gel electrophoresis. Control of the model by the computation of the reduced chi2 shows that the measurements are consistent and that electron microscopy can be used to quantify precisely the DNA deformations induced by the binding of a protein to a preferential site.

Structure-specific binding recognition of a methanogen chromosomal protein.

The archaeon Methanosarcina thermophila expresses large amounts of a small basic protein, called MC1 (methanogen chromosomal protein), which was previously identified as a DNA-binding protein possibly involved in DNA compaction in some methanogenic species. We have investigated the binding of MC1 to various kinds of branched DNA molecules whose double helix axis is severely kinked. We show that MC1 is able to distinguish and to bind preferentially to four-way junctions. This preferential binding is observed in the absence and presence of divalent cations. However, we find that MC1 has a low affinity for bulged DNA structures. These results show how MC1 is able to discriminate between different deformations of the DNA double helix.

The DNA-binding protein II from Zymomonas mobilis. Complete amino acid sequence and interaction with DNA.

The primary structure of the DNA-binding protein II from Zymomonas mobilis has been determined from data provided by automated Edman degradation of the intact protein and of peptides derived from cleavage at aspartic acid and arginine residues. When compared with the homologous protein isolated from other bacteria, the DNA-binding protein II from Z mobilis shows many substitutions. Several non-conservative substitutions at positions usually highly conserved in this type of protein probably account for the weaker DNA-binding activity of this protein compared to that of the E coli protein.

Preferential binding of the archaebacterial histone-like MC1 protein to negatively supercoiled DNA minicircles.

The interaction of the archaebacterial MC1 protein with 207 bp negatively supercoiled DNA minicircles has been examined by gel retardation assays and compared to that observed with the relaxed DNA minicircle. MC1 binding induces a drastic DNA conformational change of each minicircle, leading to an increase of the electrophoretic mobility of the DNA. A slight increase in salt concentration enhances the amount of bound MC1, and high NaCl concentrations are required to dissociate the complexes. Furthermore, the salt effect on binding depends on the supercoiling state of the DNA. The dissociation rates decrease with increasing linking difference of the minicircles relative to their relaxed configuration to reach a maximum at -2 turns. In addition, differences between the topoisomers are also observed in terms of stoichiometry of the strongest complexes. So with the -2 topoisomer the complex with two MC1 molecules is the most stable, while with the -1 and -3 topoisomers, the strongest ones are those with one MC1 molecule per DNA ring.

[Complementarity of microscopies in the structural analysis of DNA minicircles associated to protein MC1]. / Complémentarité des microscopies dans l'analyse structurale de minicercles d'ADN associés à la protéine MC1.

Electron microscopy of DNA, either free or complexed with ligands, allows the analysis of local conformational variations along individual molecules. Electron microscopy is unique, in that it has the capacity to determine the average behaviour of a population of molecules observed individually, and can thus provide a better appreciation of variability within the series of molecules than biophysical or biochemical methods. Very encouraging results have been obtained by cryoelectron and near-field microscopies, especially atomic force microscopy, in parallel with traditional techniques for visualizing DNA molecules adsorbed onto a support film. Differences in sample processing procedures and image formation modes render these 3 types of microscopies complementary. The torsional stress of a DNA molecule together with a local curvature induced by the protein MC1 from archaebacteria, can be detected within minicircles comprising 207 base pairs.

Conformational changes of DNA minicircles upon the binding of the archaebacterial histone-like protein MC1.

Binding of the archaebacterial histone-like protein MC1 to DNA minicircles has been examined by gel retardation and electron microscopy. MC1 preferentially binds to a 207-base pair relaxed DNA minicircle as compared with the linear fragment. Random binding is observed at very low ionic strength, and a slight increase in salt concentration highly favors the formation of a complex that corresponds to the binding of two MC1 molecules per DNA ring. Measurements of dissociation rates show that this complex is remarkably stable, and electron microscopy reveals that it is characterized by two diametrically opposed kinks. These results are discussed in regard to the mechanisms by which MC1 affects DNA structure.

Archaebacterial histone-like protein MC1 can exhibit a sequence-specific binding to DNA.

The binding of MC1 protein, the major chromosomal protein of the archaebacterium Methanosarcina sp. CHTI 55, to the region preceding the strongly expressed genes encoding methyl coenzyme reductase in a closely related micro-organism has been investigated. By gel retardation and DNAase I footprinting assays, we identified a preferential binding sequence in an open reading frame of unknown function. The large area of DNA protected against DNAase I is interrupted by a strong cleavage enhancement site on each strand. By circular permutation assays, we showed that the DNA bends upon MC1 binding. Furthermore we observed that the presence of a sequence outside the binding site can induce an unusual electrophoretic behaviour in some complexes.

Radiosensitivity of DNA minicircles.

DNA minicircles of 207 bp were constructed by the ligation of linear restriction fragments in the presence of various concentrations of ethidium bromide. Three topoisomers characterized by linking numbers (Lk) of 20, 19 and 18, and with helical repeats of 10.35, 10.9 and 11.5 bp/turn respectively, were obtained. They are called, respectively, relaxed minicircle or topoisomer 0, topoisomer -1 and topoisomer -2. Owing to the limited flexibility of such small circles, the stress created by the lack of 1 or 2 turns cannot be eliminated by a spatial circle-axis writhing (supercoiling) of the circular molecules. These two undertwisted, stressed topoisomers have to adopt a flat, non-crossed shape, similar to that of the relaxed minicircle. The three minicircles were irradiated with gamma-rays or fast neutrons. The same yields of single-strand breaks, double-strand breaks and alkali-induced single-strand breaks were observed for the three topoisomers showing that their base and sugar moieties are attacked equally by gamma photon- or fast neutron-induced radicals. We conclude that untwisting of a B helix does not modify the radiosensitivity of DNA.

Fluorescence study on the non-specific binding of cyclic-AMP receptor protein to DNA: effect of pH.

The binding of the cyclic-AMP receptor protein (CRP) of Escherichia coli to a non-specific DNA fragment of 46 base pairs has been studied using fluorescence spectroscopy. The equilibrium binding constant was found to be several orders of magnitude lower than in the specific binding to a DNA fragment of the same size. The salt dependence of the equilibrium binding constant indicates that the CRP makes an identical number (8) of ion pairs to this non-specific DNA fragment in the presence and absence of cAMP. This number is larger than that previously found in the specific binding process. The effect of pH on the non-specific binding was investigated. The number of ion pairs does not vary between pH 6 and 8. From the variation of the binding constant with pH it was deduced that two histidines are involved in the binding in the absence of cAMP. These are most probably the histidines 199 of each subunit. In the presence of cAMP, only one histidine participates in the binding process, indicating an asymmetric interaction between the two subunits of the CRP and the DNA.

Stoichiometry of the binding of chromosomal protein MC1 from the archaebacterium, Methanosarcina spp. CHTI55, to DNA.

We have investigated the binding stoichiometry of the chromosomal MC1 protein on DNA using the gel retardation technique. Analysis of the distribution of the complex containing 0, 1, 2, 3 ... bound proteins shows that the protein MC1 interacts with the DNA as a monomer. Binding experiments with short DNA fragments of various lengths shows that the site size is 11 bp in length. These results are compared to those obtained with other chromosomal proteins including HU protein.

Specific binding of cyclic-AMP receptor protein to DNA. Effect of the sequence and of the introduction of a nick in the binding site.

The binding of Escherichia coli Cyclic AMP Receptor Protein (CRP) to several DNA fragments of about 45 base pairs, bearing the natural lactose or galactose sites, as well as several synthetic related sites, was investigated using fluorescence spectroscopy and gel retardation experiments. The salt dependence of the equilibrium binding constant indicates that CRP makes an identical number of ion pairs with the lac, lacL8 and gal sites although the binding constants are drastically different. However increasing the symmetry of the gal site leads to an increase of the number of ion pairs between the protein and the DNA. A single strand nick was introduced at the centre of a symmetrized gal site and this reduces the binding energy of CRP by about 0.6 Kcal. These results are discussed with respect to the bending constraints imposed on the DNA by the binding of CRP. The results are in agreement with the recently published crystal structure of the CRP complexed with DNA [Schutz, S.C., Shields, G.C. and Steitz, T.A., Science 253, 1001-1007 (1991)] showing that the 90 degrees bending of the DNA in the complex results from two kinks.

The chromosomal protein MC1 from the archaebacterium Methanosarcina sp. CHTI 55 induces DNA bending and supercoiling.

We have investigated the effect on the DNA structure of protein MC1, a basic and small polypeptide (Mr 10700) representing the major chromosomal protein in Methanosarcinaceae. The ability of protein MC1 to strongly favour cyclization upon polymerization of short DNA fragments by T4 DNA ligase indicates that protein MC1 mediates DNA bending. Several negatively supercoiled topoisomers of minicircles were obtained with DNA fragments of 203 and 146 bp, their distribution depends upon the amount of protein MC1 complexed with DNA. In addition, protein MC1 can induce a compaction of a nicked plasmid.

Photochemical modification of lac repressor--III. Mutant I12X86 versus wild-type repressor.

Photodestruction of the two tryptophan (TRP) residues of the core of the wild-type Escherichia coli lac repressor has already been used as a probe in the study of interactions of the repressor with DNA and effectors. The good correlation between phenomena occurring in the core (photodestruction of TRP residues, effectors binding) and at the headpieces (DNA specific and non-specific binding) can be understood in terms of allosteric behavior of the protein. In the present study, the same approach is applied to a repressor with peculiar binding properties, the I12X86 mutant. The photodestruction of TRP residues of this tight binding repressor, bearing two different amino acids as compared to the wild-type one (Ser 61----Leu, Pro 3----Tyr) indicates a probably subtle (since not detected by classical spectroscopic methods) difference of structure of the entire protein and confirms the similarity between specific and non-specific binding of this mutant repressor to DNA, observed by other methods.

Photochemical modifications of lac repressor--II. Tryptophan photochemistry as a probe in studying the allosteric behaviour of the protein.

Irradiation of lac repressor under aerobic conditions in the near UV region (295-400 nm) decreases the Trp fluorescence of the protein. A total loss of fluorescence corresponds to the destruction of all tryptophanyl residues. Irradiation with light of wavelength between 250 and 400 nm quenches fluorescence completely when only half of the Trp residues ae destroyed. An internal photodynamic effect, in which N-formylkynurenine, a principal photoproduct of Trp, sensitizes further the destruction of the other Trp residues, accounts for our results. Experiments performed in the presence of sodium azide suggest that singlet oxygen is not involved in the destruction of Trp, but may be responsible for histidine degradation. Irradiating the repressor complexed with non-operator E. coli DNA has the same effect on Trp residues as irradiating repressor alone. On the contrary, when repressor is complexed to lac operator, both tryptophanyl residues seem to be destroyed simultaneously. This indicates that binding of specific operator DNA at the DNA site induces changes in the environment of the tryptophanyl residues (mainly tor Trp 220) which cannot further transfer in excitation energy to the photoproduct of the other Trp. A prolonged irradiation destroys the complex, leading to the same result observed for non-specific complex or for repressor alone. These results are discussed in terms of the proximity of Trp from the inducer binding site and the allosteric behaviour of the repressor.

Interaction between the cyclic AMP receptor protein and DNA. Conformational studies.

The binding of the cyclic adenosine 3',5' monophosphate receptor protein (CRP or CAP) of Escherichia coli to non-specific DNA and to a specific lac recognition sequence has been investigated by circular dichroism (c.d.) spectroscopy. The effect of cAMP and cGMP on the co-operative non-specific binding was also studied. For the non-specific binding in the absence of cAMP a c.d. change (decrease of the intensity of the positive band with a shift of its maximum to longer wavelength) indicates that the DNA undergoes a conformational change upon CRP binding. This change might reflect the formation of the solenoidal coil previously observed by electron microscopy. The amplitude of the c.d. change increases linearly with the degree of saturation of the DNA and does not depend on the size of the clusters of CRP bound. From the variation of the c.d. effect as a function of the ionic strength, the product K omega (K, the intrinsic binding constant and omega, the co-operativity parameter) could be determined. The number of ion pairs involved in complex formation between CRP and DNA was found to be six to seven. Experiments performed with several DNAs, including the alternating polymers poly[d(A-T)] and poly[d(G-C)], demonstrated that the conformational change does not depend on the DNA sequence. However, in the presence of cAMP the c.d. spectrum of the DNA shows only a small variation upon binding CRP. In contrast, in the presence of cGMP the conformational change of the DNA is similar to that observed when non-liganded CRP binds. For the specific lac operon binding, the c.d. change is different from those observed for non-specific binding in the presence or absence of cAMP. These results emphasize the high variability of the DNA structure upon binding the same protein.

Lac repressor-Lac operator complexes. Solution X-ray scattering and electrophoretic studies.

Complexes between the Lac repressor and a small DNA operator fragment (29 base pairs) were investigated using polyacrylamide gel electrophoresis and solution X-ray scattering. Titration of the DNA fragment with the repressor, followed by gel electrophoresis showed that only two types of complexes are formed with repressor/operator ratios of 0.5 and 2. Radii of gyration and forward scattered intensities were obtained from Guinier plots for repressor/operator ratios ranging from 0.3 to 2. They demonstrated that the first complex contains one repressor and two operators, whereas the second one contains four repressors and two operators. Mixing operator and repressor in equimolar concentrations leads to a mixture of both complexes. A possible model for the four repressor/two operator complex is proposed.

Ultraviolet radiation-induced derepression of the lactose operon of E. coli.

The effect of ultraviolet irradiation of a regulatory protein, the lac repressor, on its interactions with operator DNA is investigated by spectroscopic and electrophoresis methods. A second set of experiments is performed to assay the capacity of the system containing the irradiated repressor to be induced by IPTG. The protein-nucleic acid interactions are modified upon ultraviolet irradiation of the repressor. The inducer becomes ineffective and repressor stays "locked" to DNA in conditions in which the native repressor is released from the system. These facts are discussed in terms of genes repression and of promotion step in ultraviolet induced carcinogenesis.