In vitro binding of N-acetoxy-N-2-acetylaminofluorene to DNA in chromatin.

The binding of N-acetoxy-N-2-acetylaminofluorene to DNA in native and partially dehistonized chicken erythrocyte chromatin was studied. The amounts of carcinogen bound to DNA were measured, after removal of proteins with phenol, by using the absorption ratio A305/A260 or by counting the radioactivity of 14C-labeled carcinogen. Measurements of uncovered zones of DNA in chromatin were made by comparison of results obtained with free DNA and with chromatin at increasing ratios of carcinogen/nucleotide. The proportion of DNA accessible to the carcinogen was found to be 15% in native chicken erythrocyte chromatin and about 22% in native calf thymus chromatin. The amount of accessible DNA increases to 55% in chicken erythrocyte chromatin depleted of histones H1 and H5. Formaldehyde unwinding performed on DNA extracted from chromatin after modification showed an increasing number of defects in the double helix with the amount of DNA-fixed carcinogen. At high ratios of carcinogen/nucleotide, the recoveries of DNA (by phenol method) and of histones (by acidic extraction) decreased with increasing ratios. This suggests a covalent linkage between proteins and DNA.

Secondary structural modifications as a consequence of in vitro acetylation and phenanthrylation of DNA by the ultimate carcinogen N-acetoxy-N-2-acetylaminophenanthrene.

The acetic acid ester of the proximate carcinogen N-hydroxy-N-2-acetylaminophenanthrene was reacted in vitro with native and heat-denatured calf thymus DNA under various conditions. We showed that besides the phenanthrylation of the DNA bases there is an acetylation reaction of the DNA during its reaction with the ultimate carcinogen. Heat-denatured DNA is 5 to 10 times more acetylated than native DNA. This result suggests that most of the acetylation sites are nonreactive in the double-helical structure of DNA. On the other hand, the phenanthrylation of the bases is shown not to depend on the DNA secondary structure, suggesting that the phenanthrylation sites of the bases are accessible in the grooves of the DNA double helix. The influence of the DNA dynamic structure on the reactions of acetylation and phenanthrylation has been investigated by increasing the ionic strength of the incubation buffer. The melting temperature of different DNA samples, which have been reacted with different concentrations of N-acetoxy-N-2-acetylaminophenanthrene, decreases as the extent of the DNA modifications increases. This thermal destabilization of the double helix is tentatively attributed to the phenanthrylation rather than to the acetylation reaction.

Interaction between hydroxystilbamidine and DNA. I. Binding isotherms and thermodynamics of the association.

Isotherms describing the binding of hydroxystilbamidine to DNA and polydeoxyribonucleotides were obtained by means of sedimentation or dialysis experiments and fluorescence measurements, over a large range of ionic strengths, temperatures and base compositions. Two different sets of binding sites are necessary to explain the shapes of the isotherms. The first one is characterized by a higher binding constant, a topological specificity for the A-T pair, exclusion of four base pairs per bound dye molecule, the involvement of two ion-pairs, an almost purely entropic free energy of binding and a large enhancement of the blue fluorescence (450 nm) when the site corresponds to three adjacent A-T pairs. The latter does not present any specificity nor enhancement of fluorescence and only one ion-pair is formed. From the geometry of the dye and its selective binding to a double stranded structure, the hydroxystilbamidine molecule in the first set of sites is likely to be situated in the small groove astride the two complementary strands and slightly distorting the helical structure. The angle of the dye axis with the helix axis has a value close to 47 degrees. No definite explanation could be given for the specific binding of hydroxystilbamidine but the phenolic hydroxyl group is likely to play a major role. The hydroxystilbamidine molecule can be considered as a useful tool for checking the accessibility of the small groove.

Interaction between hydroxystilbamidine and DNA. II. Temperature jump relaxation study. Dynamics of nucleic acids and polynucleotides.

A temperature-jump relaxation study of the interaction of hydroxystilbamidine with DNA and synthetic polynucleotides has been performed. Two concentration dependent relaxation times tau1 and tau2 have been observed in the submillisecond range when detecting relaxation effects by means of light absorption. The longer of these two times (tau1) is also observed when using "blue" or "red" fluorescence detection. In the longer time scale the "red" fluorescence shows no other relaxation but the blue fluorescence shows two additional relaxation processes (tau3 and tau4) which correspond to an increase of fluorescence with temperature and which are independent of concentration. The experimental results clearly indicate that tau1 and tau2 are associated with the binding of the dye to strong and weak binding sites, respectively. A kinetic model is given to explain the results. It allows the determination of the four rate constants for the two binding reactions and yields equilibrium association constants in good agreement with those obtained from stoichiometric studies. The study of the effect of temperature, nature of the polymer, ionic strength and fraction of bound dye on tau3 and tau4 indicates that the dye acts only as a "blue" fluorescence probe of some processes involving the DNA or polynucleotide alone. These processes appear to be related with the dynamic structure of the polymers.

Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.

Comparison between histones FV and F2a2 of chicken erythrocyte. I. Structure, stability and conformation of the free proteins.

Purified chicken erythrocyte histones FV and F2a2 were studied by means of circular dichroism as a function of ionic strength and temperature. The percentage of alpha-helical regions was calculated by comparison with reference spectra obtained with four standard proteins of known tertiary structure. Maximal alpha-helical organization, reached in high ionic strength, was estimated to 14% and 23% for FV and F2a2 respectively. We have compared our experimental determinations of the secondary structure of F2a2 with predictions made from amino-acid sequence according to Fasman's rules. When instability induced by the presence of charged residues close together is taken into account, a good agreement is found between predicted and observed values. The thermal denaturation of FV is cooperative and, unlike F2a2, seems to obey a two-state transition. The classical Arrhenius plot is linear, which indicates that the heat capacity is the same in both the native and the denatured state. Such a behaviour is typical of an expanded configuration of FV even in the "native" state.

The secondary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by circular dichroic spectroscopy.

Ribosomal proteins from Escherichia coli MRE600 have been obtained by a new, mild purification procedure. This involves extraction of the subunits with salt followed by chromatographic fractionation in the presence of salt. The use of urea or other denaturing agents and conditions is avoided. A survey of the secondary structure of the 30 S and 50 S proteins, as observed by circular dichroic spectroscopy, is presented. The spectra have been analysed by a new procedure which uses a library of 16 circular dichroic spectra of proteins with a known three-dimensional structure. This method provides a more reliable analysis, especially of the contribution from beta-sheet. The results show that most of the 30 S proteins have a high alpha-helix content, whereas the 50 S proteins are more diverse. The latter group shows a larger contribution from beta-sheet. The data presented here are compared with those already published for a number of proteins which were, with one exception, prepared in the presence of urea. In most cases we find higher alpha-helix and beta-sheet values for the salt-extracted proteins than for the corresponding urea-treated proteins. In those cases, however, where special care was taken to renature the urea-treated proteins agreement is found to within the expected experimental error. The results show that salt-extracted ribosomal proteins have a well-defined secondary structure with a relatively small contribution from unordered structure.

Co-operativity value of DNA RecA protein interaction. Influence of the protein quaternary structure on the binding analysis.

We show that an erroneous estimation of the quaternary structure of free protein distorts the quantitative analysis of its interaction with DNA, affecting especially the co-operativity value found. This could explain the discrepancy reported for the co-operativity value of the RecA-DNA interaction. The large cluster observed by electron microscopy indicates a very high co-operativity, whereas analysis of the binding isotherm indicates a moderate one, on the assumption of monomer. But if RecA is a large oligomer, the latter analysis would give a much higher co-operativity value and the former a smaller one, and they would be in accordance. Our sedimentation and light-scattering experiments suggest an oligomerization of about 30-mer or more, and support this explanation.

Specificity of N-acetoxy-N-2-acetylaminofluorene-induced frameshift mutation spectrum in mismatch repair deficient Escherichia coli strains mutH, L, S and U.

The mismatch repair system of Escherichia coli is known to contribute to the fidelity of the replicational process. This system involves the functions of mutH, mutL, mutS and mutU (uvrD) loci which recognize mispaired bases as a consequence of errors due to the polymerase itself. Chemical modifications of DNA have also been suspected to create mispaired bases which, if the mispaired bases are removed, will lead to mutations by frameshift. Using the pBR322 plasmid DNA modified by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) we have investigated this possibility in a forward mutational assay (tetracycline sensitivity). This fluorene derivative has been shown to induce predominantly frameshift mutations. Our results show that: The sensitivity of the deficient strains mutH, mutL and mutS to the AAF adducts is similar to that of the corresponding wild-type strain. However, the mutU strain appears much more sensitive to those adducts although less than a uvrA, B or C-deficient strain. This suggests that the mutU gene product is involved in the repair of AAF adducts. For the four mut deficient strains, and as it was shown with the wild-type strain, AAF adducts induced mutations to tetracycline sensitivity are only observed when the SOS system of the host bacteria is induced by irradiation of the cells prior to transformation with the modified plasmid. The mutation frequencies depend upon the ultraviolet light doses and similar maxima were found for the four mut strains and the corresponding wild-type strain. In agreement with the results obtained with wild-type or uvrA strains we observe that AAF adducts induce mostly frameshift mutations in the mut strains. Two types of hot spots of mutagenesis were described in wild-type and uvrA strains occurring either at repetitive sequences or at sequences of the type 5' G-G-C-G-C-C 3' (NarI restriction enzyme recognition sequence). While the second type of mutational hot spot does exist in the mismatch repair-deficient strains, we observe that the repetitive sequences are no longer hot spots of mutations in these strains, suggesting that the mismatch repair protein complex is involved in the establishment of AAF-induced frameshift mutations at repetitive sequences.

Promoter properties and negative regulation of the uvrA gene by the LexA repressor and its amino-terminal DNA binding domain.

A comparative study of the interaction of the LexA repressor of Escherichia coli and of its amino-terminal DNA binding domain to the uvrA operator has been undertaken. Most of the binding constants are determined from competition experiments with RNA polymerase by measuring the time-course of the abortive initiation transcriptional activity. The presence of repressor increases the lag time, tau, without affecting the final maximum activity. The inhibition of transcription by LexA, at least in the case of the uvrA gene, is thus a transient, time-dependent phenomenon, because once the RNA polymerase is engaged in a stable "open" complex, it is quasi-irreversibly trapped in this state. A study of the binding constants as a function of ionic strength suggests the formation of 5.5(+/- 1) salt bridges between the uvrA operator and a LexA dimer. Surprisingly, the binding affinity of the amino-terminal domain was only about one order of magnitude smaller than that of the entire LexA repressor. The determination of the binding constant of the RNA polymerase to the "closed" uvrA promoter (KB approximately 1 X 10(7) to 2 X 10(7) M-1) allowed us to determine theoretical repression curves for the two repressor species. These calculations show that the binding constant found for LexA is sufficiently high to account for substantial or complete repression, and that of the amino-terminal domain is sufficiently low to account for partial or nearly full induction. Under solvent conditions used by others for the determination of binding constants to other SOS operators by DNAase I footprinting, the uvrA operator turns out to be a rather weak one (K approximately 3 X 10(7) M-1), being comparable with that of the uvrB gene. The uvrA promoter is "association-limited" with a KB X k2 product fitting very nicely the homology score for the promoter of 55.

Histone phosphorylation in native chromatin induces local structural changes as probed by electric birefringence.

In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.

Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences.

Forward mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) in the tetracycline resistance gene carried on plasmid pBR322 are shown to be dependent upon the induction of the host SOS functions in wild-type and umuC Escherichia coli cells. The mutation frequency in the umuC strain is equal to about 40% of the mutation frequency observed in the umu+ background. In the excision-repair-deficient uvrA mutant strain the mutagenic response is the same as in SOS-induced wild-type cells whether or not the uvrA bacteria are SOS-induced. Equal mutation frequencies are obtained in both the wild-type and the uvrA strains for equal modification levels although the survival of AAF-modified plasmid DNA is greatly reduced in the uvrA strain as compared to the wild-type strain. Sequence analysis of the mutations reveals that more than 90% of the N-Aco-AAF-induced mutations are frameshift mutations. Two types of mutational hotspots are observed occurring either at repetitive sequences or at non-repetitive sequences. Both types of mutants appear at similar locations and frequencies in both the wild-type and the uvrA strains. On the other hand, only the non-repetitive sequence mutants are obtained in the umuC background. These non-repetitive sequence mutants preferentially occur within the sequence 5' G-G-C-G-C-C 3' (the NarI restriction enzyme recognition sequence). The analysis of the -AAF binding spectrum to the same DNA fragment shows that there is no direct correlation between the modification spectrum and the mutation spectrum. We suggest that certain sequences are "mutation-prone" in the sense that only these sequences can be efficiently mutated as the result of an active processing mediated by specific proteins. When a sequence is said to be mutation-prone it probably corresponds to a particular structure that is induced within this sequence as a result of the binding to the DNA of the mutagen. This sequence-specific conformational change is the substrate for the protein(s) that fixes the mutation. The mutagenic processing pathway(s) is part of the cellular response to DNA-damaging agents (the so-called SOS response). Two pathways for frameshift mutagenesis are suggested by the data: an umuC-dependent pathway, which is involved in the mutagenic processing of lesions within repetitive sequences; an umuC-independent pathway responsible for the fixation of mutations within specific non-repetitive sequences.

Cooperative and salt-resistant binding of lexA protein to non-operator DNA.

The interaction of the lexA repressor of E. coli with poly[d(A-T)] has been studied by circular dichroism. The binding induces an about 2-fold increase of the circular dichroism intensity at 263 nm, pointing out a conformational change of the nucleic acid. The observed spectral changes are very similar to those observed for the binding of the lac repressor to poly[d(A-T)] and natural DNA. At elevated ionic strength the binding isotherms do show a pronounced sigmoidal shape indicating a cooperative mode of binding.

Fluorescence study of the RecA-dependent proteolysis of LexA, the repressor of the SOS system in Escherichia coli.

The fluorescence of the LexA protein, the common repressor of the SOS system in Escherichia coli decreases by about 30% upon incubation with the RecA protein, and its cofactors ATP [or its non-hydrolysable analogue adenosine-5'-O-(3-thiotriphosphate), ATP gamma S] Mg2+ and single-stranded DNA. In the absence of any one of these elements required for the RecA-dependent proteolysis of LexA, this fluorescence change was not observed. The final fluorescence change depends only upon the concentration of LexA regardless of that of RecA. The time course of the fluorescence decrease corresponds well with the kinetics of the decrease of intact LexA protein and the increase of its 2 proteolytic fragments as determined by SDS-polyacrylamide gel electrophoresis. These results allow us to use the fluorescence change as a signal for a detailed kinetic analysis. The velocity of the proteolysis (d[LexA]/dt) is proportional to the concentration of LexA and RecA indicating that the formation of the LexA-RecA complex is the limiting step.

Fast abortive initiation of uvrA promoter in a supercoiled plasmid studied by stopped-flow techniques.

In order to follow the fast kinetics of abortive initiation (lag time from 1 ms to 10 s), we have built a stopped-flow apparatus equipped for fluorescence detection. The small volume used for each assay (35 microliters), and the short dead time (approximately 0.5 ms) are the essential advantages of this apparatus. Supercoiling of DNA affects considerably the initiation of transcription from the uvrA promoter. It decreases the lag time due to the isomerisation process 3-fold. Nevertheless, it does not change significantly the product KBk2, which is indicative of promoter strength and shows that uvrA is an 'association-limited' promoter. The presence of the LexA repressor increases the lag time considerably. At least for small RNA polymerase concentrations this increase is stronger for supercoiled than for linearized DNA.

Fluorescence anisotropy decay of ethidium bromide bound to nucleosomal core particles.

We have measured the fluorescence anisotropy decay of ethidium bromide bound to nucleosomal core particles (145 DNA base pairs) for very small values of the binding ratio (0.0005 less than or equal to r less than or equal to 0.01). For r = 0.0005 the anisotropy decay could be described by a sum of two exponential functions. The two correlation times theta 1 and theta 2 increase with r until r congruent to 0.0025 and then decrease while the apparent fundamental anisotropy A'0 decreases until r congruent to 0.0025 and then remains constant. The anisotropy decay parameters of the first ethidium molecule bound to a core particle have been obtained by extrapolating theta 1, theta 2 and A'0 to r = 0. We propose the following interpretation of these results. The first bound ethidium molecule is located on a DNA segment linked by its two ends to the histone core. This ethidium molecule follows the torsional motion of the DNA segment. The length of this segment (15 base pairs) was determined by fitting a mathematical expression, derived from the torsional dynamics of DNA, to the extrapolated anisotropy decay. The second ethidium molecule binds to the same DNA segment which explains the decrease of A'0 by fast excitation energy transfer. At the same time theta 1 and theta 2 increase. On binding, a third ethidium molecule breaks the links between the DNA segment and the histone core. This entails the decrease of theta 1 and theta 2.

Analysis at the sequence level of mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene.

The covalent binding of an ultimate carcinogen to the DNA bases or phosphate groups creates a premutational lesion that in vivo is processed by the repair, replication and recombination enzymes, and eventually may be converted into a mutation. Being interested in the way that an initial premutational event is converted into a stable heritable mutation, we have sequenced stable mutations in a gene that has formed covalent adducts in vitro with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF, a model for the ultimate metabolite of the rat liver carcinogen 2-acetylaminofluorene, AAF). In vivo studies have shown the mutagenicity of AAF and its derivatives in both bacterial and eukaryotic systems. N-AcO-AAF reacts in vitro with DNA leading mainly to the formation of a guanine adduct, N-2-(deoxyguanosin-8-yl)-acetylaminofluorene (80%) and to at least three minor adducts. Studies by our group showed that binding of N-AcO-AAF to DNA resulted in a local distortion of the DNA helix around the C-8 adduct (the insertion-denaturation model). We describe here the analysis of forward mutations induced in the tetracycline-resistance gene of pBR322 by directing the chemical reaction of the carcinogen to a small restriction fragment (BamHI-SalI) inside the antibiotic-resistance gene. Mutants are selected for ampicillin (Ap) resistance and tetracycline (Tc) sensitivity. The plasmid DNA of such mutants was analyzed for sequence changes in the fragment where the AAF binding had been directed. We show here that the mutations are mainly frameshifts involving GC base pairs and that certain base pairs (hotspots) are affected at high frequencies.

Effect of tetramethylammonium ions on conformational changes of DNA in the premelting temperature range.

The reversible conformational change of DNAs and polydeoxyribonucleotides occurring before melting was followed by circular dichroism. deltatheta/deltaT, the rate of change of ellipticity theta with temperature, was used mainly as a measure of this premelting phenomenon. If sodium ions were replaced by tetramethylammonium ions deltatheta/deltaT decreased for poly (dA) poly (dT) and poly (dA.dT) poly (dT.dA), but increased for poly (dG.dC) poly (dC.dG). DNAs of different base composition showed no more premelting (deltatheta/deltaT approximately 0) even at low molarities of TMACl provided the Na/TMA ratio was very small. For all cases studied the theta values at 0 degrees C and at a given ionic strength were smaller in NaCl than in TMACl. When studying the series of ammonium ions from NH4+ to (C2H5)4N+, the deltatheta/deltaT values first decreased, going through zero with TMA+ ions, and then increased again. A tentative and qualitative explanation of our results can be given: (a) Hydration of the polymers increases in presence of TMA ions and their average stability decreases; locally, however, (AT) pairs are preferentially stabilized by TMA ions owing to a specific interaction at the level of O2 of thymine. (b) In order to explain the different behaviour of (AT) polymers and DNA, it is assumed that only the B structure is able to accommodate TMA ions in the small groove of the double stranded helix.

Core particle stability critically depends upon a small number of terminal nucleotides.

An apparently homogeneous population of core particles is in fact composed of three subpopulations which behave differently when exposed to a high concentration of ethidium bromide or to 0.6 M NaCl. These subspecies have been identified by the use of several techniques, viz., electron microscopy, sedimentation velocity and circular dichroism. The electrophoretic analysis of their DNA leads to the conclusion that core particle stability critically depends upon a small number of terminal nucleotides.

Thermal transition of core particle is not a two-state process.

Thermal transition of core particle which occurs before melting of DNA and can be followed by circular dichroism is not a two-state process; it is the result of two processes which cannot be dissociated in static experiments: unfolding of core particles is immediately followed by their aggregation. It is thus impossible to get thermodynamic parameters of core particle unfolding from its thermal transition monitored by circular dichroism. Thermal denaturation kinetics of core particles gives some information about their stability. Finally core particle structure is more stable in chromatin than in its isolated state.