Radiolysis of nucleosome core DNA: a modelling approach

Purpose : To calculate the expected pattern of frank strand breaks (FSB) induced in the DNA of a nucleosome core particle (NCP) by the attack of radiolytic OH ?radicals and to compare this with the experimental pattern of FSB induced by the in vitro irradiation of chicken NCP. Materials and methods : The structure of NCP was obtained from the PDB databank. Using molecular modelling, the structure of a linear DNA fragment with the central 60 bp sequence of NCPDNA was determined. The accessibility of the sugar attack sites to OH ?radicals at each nucleotide of the linear fragment or of the NCP-DNA was calculated. The probability of reaction of OH ?with each sugar reactive site was calculated using a MonteCarlo method-based stochastic model. Results : The accessibility of attack sites in the NCP-DNA and the calculated probabilities of sugar attack are mainly modulated through masking by histones, and only slightly through bending. The periodicity of the calculated FSB probabilities is identical to that of the experimental FSB probabilities in chicken NCP (period 10.4 ±0.1bp). Conclusions : The good agreement between the calculated and experimental results recommends the presented modelling procedure as a tool in predicting the radiosensitivity of DNA in DNA-ligand complexes of known structure.     

Monte Carlo simulation of radiolytic attack to 5'-d[T4G4]4 sequence in a unimolecular quadruplex.

PURPOSE: To extend to a quadruplex the stochastic model of radiolytic attack previously applied to a quasi-random duplex DNA. MATERIALS AND METHODS: The quadruplex structure is obtained from the PDB databank (first structure from 201D entry). The probabilities of OH* radical attack at all sugar and base reaction sites are calculated using a stochastic model based on the Monte Carlo method. RESULTS: Good agreement between the calculated and experimental frank strand break (FSB) probabilities is obtained using the relative efficiencies of conversion of the C4' and C5'-centred radicals into FSB determined for the quasi-random duplex (2.8:1 respectively). Efficiencies of base radicals-to-alkali-revealed breaks (ARB) conversion are determined by fitting the calculated probabilities of base attack to the previously reported experimental probabilities for ARB. The efficiency of conversion of thymine radical into an ARB is 3.4+/-0.7 times higher than for guanine radical. CONCLUSIONS: This paper supports the calculation method and allows evaluation of the relative efficiencies of thymine and guanine radicals-to-ARB conversion.     

Radioprotection of DNA by spermine: a molecular modelling approach.

PURPOSE: To observe and explain the sequence-dependence of DNA radioprotection by spermine. MATERIALS AND METHODS: Sequencing gel electrophoresis was used to analyse the probability of frank strand break (FSB) induction at each nucleotide site. Molecular modelling of complexes of DNA with spermine molecules and of a curved electrically null DNA has been performed. RESULTS: The effect of spermine on radiation-induced strand breakage varied significantly along the studied fragment. At low spermine concentration, some sequences were protected while others were unprotected. Molecular modelling calculations show that the most electro-negative sites are located in the minor or in the major groove of DNA. The positively charged spermine (Z=+4) should preferentially bind to such sites. When bound in the minor groove, spermine triggers a reduction of the accessibility of radiolytic attack sites to OH* radicals. This is due to induced structural modifications and to the masking of attack sites. In the case of major groove binding, no reduction of accessibility occurs. This type of binding can explain the lack of protection of sequences with electro-negative sites in the major groove. At high spermine concentration, the fragment is strongly protected. A nucleosome-like pattern of breakage with periodically distributed regions of protection was observed. Molecular modelling calculations show that the accessibility of the attack sites in a curved electrically null DNA is also periodically reduced. CONCLUSIONS: Molecular modelling of DNA-spermine complexes that takes into account the electrostatic properties of DNA, allows an explanation of the experimentally observed effects of spermine on DNA radiosensitivity.     

Radioprotection of DNA by spermine: a molecular modelling approach

Purpose: To observe and explain the sequence-dependence of DNA radioprotection by spermine. Materials and methods: Sequencing gel electrophoresis was used to analyse the probability of frank strand break (FSB) induction at each nucleotide site. Molecular modelling of complexes of DNA with spermine molecules and of a curved electrically null DNA has been performed. Results: The effect of spermine on radiation-induced strand breakage varied significantly along the studied fragment. At low spermine concentration, some sequences were protected while others were unprotected. Molecular modelling calculations show that the most electro-negative sites are located in the minor or in the major groove of DNA. The positively charged spermine (Z4) should preferentially bind to such sites. When bound in the minor groove, spermine triggers a reduction of the accessibility of radiolytic attack sites to OH radicals. This is due to induced structural modifications and to the masking of attack sites. In the case of major groove binding, no reduction of accessibility occurs. This type of binding can explain the lack of protection of sequences with electro-negative sites in the major groove. At high spermine concentration, the fragment is strongly protected. A nucleosome-like pattern of breakage with periodically distributed regions of protection was observed. Molecular modelling calculations show that the accessibility of the attack sites in a curved electrically nullDNA is also periodically reduced. Conclusions: Molecular modelling of DNA-spermine complexes that takes into account the electrostatic properties of DNA, allows an explanation of the experimentally observed effects of spermine on DNA radiosensitivity.     

Radiation damage to triplex DNA induced by gamma-rays: a footprinting study and Monte Carlo simulation

PURPOSE: This study is aimed at comparing the radiosensitivity of a 21-mer DNA fragment in duplex and triplex form (Py x Pu duplex and Py x Pu:Py triplex) and to give insights into the fine structural features due to the different strandedness. MATERIALS AND METHODS: The triplex sample was characterized by means of electrophoretic mobility, circular dichroism spectra and UV melting experiments. Triplex and duplex, labelled on the homopyrimidine or homopurine strand, were irradiated with 60Co gamma-rays. The samples were analysed by sequencing gel electrophoresis and the patterns of relative probabilities of frank strand breakage (FSB) and alkali-revealed breakage (ARB) at each nucleotide site were determined. Relative probabilities of OH* radical attack to duplex and triplex DNA built up with a molecular modelling software were calculated using a previously reported simulation procedure (Sy et al. 1997). The experimental data were compared with the results of the theoretical simulations of OH* radical attack to DNA. RESULTS: Globally, the duplex is more attacked than the triplex, mainly in the Pu strand. Sequence-dependent variations of FSB and FSB+ARB probabilities in both duplex and triplex were revealed. No significant differences between the patterns of damage in the triplex and in the duplex were observed. CONCLUSIONS: The presence of the third Py strand located in the major groove of the DNA duplex modifies the total yields of radiation-induced DNA damage, but not the sequence-dependent patterns of relative probability of damage at each nucleotide site.     

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 OH ? 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.     

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.     

Radiation damage to triplex DNA induced by γ-rays: a footprinting study and Monte Carlo simulation

Purpose : This study is aimed at comparing the radiosensitivity of a 21-mer DNA fragment in duplex and triplex form (Py?Pu duplex and Py?Pu:Py triplex) and to give insights into the fine structural features due to the different strandedness. Materials and methods : The triplex sample was characterized by means of electrophoretic mobility, circular dichroism spectra and UV melting experiments. Triplex and duplex, labelled on the homopyrimidine or homopurine strand, were irradiated with 60 Co γ-rays. The samples were analysed by sequencing gel electrophoresis and the patterns of relative probabilities of frank strand breakage (FSB) and alkali-revealed breakage (ARB) at each nucleotide site were determined. Relative probabilities of OH? radical attack to duplex and triplex DNA built up with a molecular modelling software were calculated using a previously reported simulation procedure (Sy et al. 1997). The experimental data were compared with the results of the theoretical simulations of OH? radical attack to DNA. Results : Globally, the duplex is more attacked than the triplex, mainly in the Pu strand. Sequence-dependent variations of FSB and FSB + ARB probabilities in both duplex and triplex were revealed. No significant differences between the patterns of damage in the triplex and in the duplex were observed. Conclusions : The presence of the third Py strand located in the major groove of the DNA duplex modifies the total yields of radiation-induced DNA damage, but not the sequence-dependent patterns of relative probability of damage at each nucleotide site.     

Effect of ethidium bromide intercalation on DNA radiosensitivity

PURPOSE: To assess the influence of the intercalating drug ethidium bromide (EtBr) on the yields of single strand breaks (ssb) induced by fast neutrons in supercoiled pBR322 plasmid and in a linear DNA restriction fragment. MATERIALS AND METHODS: The yield of ssb in the plasmid was measured by agarose gel electrophoresis. The proportion of fragments bearing one ssb and the probability of breakage at each nucleotide site was determined using sequencing gel electrophoresis. The volume variations due to the intercalation of EtBr were calculated. The expected radio-modifying effect at each nucleotide site of the linear fragment was evaluated using a reported simulation procedure. RESULTS: The ssb yield in the plasmid increased for concentrations up to 0.04 drug/bp and fell back in the range 0.04-0.1 drug/bp. For the linear DNA, only a slight protective effect was observed over the whole concentration range. The effect was almost the same at all nucleotide sites. CONCLUSION: For the linear DNA fragment, radioprotection was mainly due to scavenging of OH* radicals by the intercalated drug. For the plasmid, the radio-modifying effect results mainly from the variation of its effective volume, due to the modification of superhelicity.     

Hydroxyl radical scavenging by carnosine and Cu(II)-carnosine complexes: a pulse-radiolysis and spectroscopic study.

PURPOSE: To obtain a wider insight into the general properties of carnosine and to provide support to its anti-oxidative role. This property is hypothesized to be linked to various mechanisms including free-radical scavenging and metal chelation (i.e. Cu(II)). METHODS: Pulse-radiolysis experiments were performed by a 12 MeV electron linear accelerator (LINAC) on carnosine/copper(II) (2:1) and carnosine aqueous solutions at different pH. Raman spectra of solid samples were obtained by a Bruker IFS 66 spectrometer. RESULTS: As well as for free carnosine, in the presence of copper ions the interaction of carnosine with *OH radicals involves the imidazole group of the molecule. The oxidation of copper (II)-carnosine system by *OH radicals is related to the pH-dependent structure of the copper(II)-carnosine complex. Raman spectra indicate that at alkaline pH the formation of a dimeric species containing two carnosine molecules complexed to two Cu2+ ions takes place. This structure can address the *OH attack more selectively than carnosine itself to different sites of the imidazole ring. The formation of at least two different *OH-radical adducts occurs and positions C(2) and C(5) of the imidazole ring are the preferential sites for the *OH attack, as the heterocyclic ring is mainly present as its N(1)-protonated tautomeric form. CONCLUSION: These studies provide further evidence about the formation of carnosine-copper complexes and the predominance of a dimeric structure at slightly basic pH. The chelation of Cu(II) is not detrimental to the scavenging ability of carnosine. Raman spectra are helpful in identifying the structure of the copper(II)-carnosine complexes and in predicting the preferential sites for the *OH attack to the carnosine-copper system.     

Calculated strand breaks from 125I in coiled DNA

Purpose: DNA single strand breaks (SSB) and double-strand breaks (DSB) induced by Auger electrons from incorporated ¹²⁵I decay were calculated using a B-DNA model to assess contributions from direct and OH damage and effects of higher-order structure. Three decay sites, linker DNA, nucleosome, and two adjacent nucleosomes, were assessed and compared to experimental data.

Method: A Monte Carlo track structure code for electron was used to track electrons, OH and H radicals through linear and a higher-order model of B-DNA. Direct and indirect DNA hits were scored and used to determine SSB and DSB.

Results: The three different ¹²⁵I decay locations produced different number of DSBs and fraction of radical damage. The average number of DSB per ¹²⁵I decay was 0.83, 0.86 and 1.33, respectively, for the three sites. OH radical attack contributed to or exclusively caused 70%, 57%, and 50%, of the DSBs located in the entire model. When only 10 base pairs on either side of the incorporation site were considered, radical damage contributions were 40%, 25% and 67%, respectively. Locations distant from the site of incorporation, however, consistently yielded 70–80% of the DSB from radical attack.

Conclusions: Coiling of DNA can greatly change both the absolute number of DSB per incorporated ¹²⁵I decay and the relative contributions of radical damage to the local site of decay and, to a lesser extent, the average over all DNA. Higher order structure only slightly affects the number and quality of DNA damage to distant locations, which is mostly from radical attack.     

Modelling study on the protective role of OH radical scavengers and DNA higher-order structures in induction of single- and double-strand break by gamma-radiation

PURPOSE: To quantify the protective effects of (non-histonic) OH-radical scavengers and DNA higher-order structures in induction of single- (ssbs) and double-strand breaks (dsbs) by gamma-rays. MATERIALS AND METHODS: Spatial distributions of energy depositions by gamma-rays in liquid water were modelled with the track structure modules of the biophysical simulation code PARTRAC. Such distributions were superimposed on different DNA structure models (e.g. linear DNA, SV40 'minichromosomes' and compact chromatin), and direct energy depositions in the sugar-phosphate were considered as potential (direct) ssbs. The diffusion and interaction of the main chemical species produced in liquid water radiolysis were explicitly simulated, and reactions of *OH with the sugar-phosphate were considered as potential (indirect) ssbs. Two ssb on opposite DNA strands within 10 base pairs were considered as one dsb. Yields of ssb and dsb Gy(-1) Dalton(-1) in different DNA target structures were calculated as a function of the *OH mean lifetime, whose inverse value was taken as representative of the scavenging capacity of the DNA environment. RESULTS AND CONCLUSIONS: A further validation of the models implemented in the PARTRAC code has been provided, thus allowing a better understanding of the mechanisms underlying DNA damage. More specifically, the protection due to *OH scavengers was separately quantified with respect to that due to histones and chromatin folding, which could be 'switched off' in the simulations. As expected, for a given value of the environment scavenging capacity, linear DNA was more susceptible to strand breakage than SV40 minichromosomes, which in turn showed higher damage yields with respect to cellular DNA due to the larger accessibility offered to *OH. Furthermore, by increasing the scavenging capacity, the break yields decreased in all structures and tended to coincide with direct damage yields. Very good agreement was found with available experimental data. Comparisons with data on 'nucleoid' DNA (i.e. unfolded and histone-depleted DNA) also suggested that the experimental procedures used to obtain such structures might lower the environment scavenging capacity owing to the loss of cellular scavengers.     

DNA radiolysis in DNA-protein complexes: a stochastic simulation of attack by hydroxyl radicals

PURPOSE: To propose an improved version of RADACK, a stochastic simulation of radiolytic attack on DNA, that takes into account the reactivity of each amino acid of a specifically bound protein with hydroxyl radicals. To apply it to the natural lactose operator-repressor complex taking advantage of recently reported structures. To compare the obtained probabilities of DNA strand break induction with those calculated with the previous versions and with an experimental pattern of strand break probabilities. MATERIALS AND METHODS: Models of complexes close to the natural ones, derived from crystallography- and NMR-based structures recently available in the PDB databank, were used. The specific chemical reactivity of each amino acid was introduced in the new version of RADACK (the reactivity model). The probabilities of strand break induction by the irradiation of the complex were calculated with this new version as well as with previous ones. RESULTS: The patterns of probabilities of strand break induction calculated with the improved version of RADACK were partially different from those obtained with previous versions. The patterns obtained for both, using putative models of natural complexes, were consistent with the experimental results, but some discrepancies were suggestive of slight differences between these structures and the real natural system. The crystallographic structure agreed best with the experimental results. CONCLUSIONS: A new version of RADACK was validated that took into account the reactivity of atoms in both DNA and protein. The putative modelled structures of a natural lactose operator-repressor complex were discussed.     

Influence of an 8-oxoadenine lesion on the structural and dynamic features of a 30-mer DNA fragment with and without a mismatch.

PURPOSE: To elucidate the influence of the oxidative lesion 7,8-dihydro-8-oxoadenine (8-oxoA) on the structural and dynamic features of a 30-mer DNA fragment, and to understand if differences occur when C is positioned opposite 8-oxoA instead of T. MATERIALS AND METHODS: Two 30-mer DNA oligomers with or without the 8-oxoA and two complementary oligomers with C or T base opposite the lesion site were synthesized and annealed. Duplexes named AT, A*T, AC and A*C were characterized by means of circular dichroism and UV denaturation measurements. gamma-Ray footprinting experiments were performed to give insight into their fine three-dimensional structure. Elastic torsional constants were derived by following the decay of the fluorescence polarization anisotropy (FPA) of the ethidium-DNA complexes measured by multifrequency-phase fluorometry. RESULTS: The introduction of one oxidative lesion in a 30-mer DNA oligomer with and without a mismatch did not cause relevant changes in their overall conformation and slightly modified their elastic properties. Small energetic differences were revealed by thermodynamic analysis in the sample bearing both the oxidative lesion and the mismatch. Minor variations in the cleavage pattern due to the hydroxyl radicals in the A*T sample were observed and present along the entire DNA fragment length. In the A*C sample, by contrast, there was a major modification in the cleavage pattern extending for about 11 bases around the lesion, especially towards the 5'-end. CONCLUSIONS: Differences in the fine structure and in the elastic properties between the A*T and A*C samples were observed, while their overall conformation was unchanged. The results are consistent with the hypothesis that the observed local changes of the double helix structure in A*C are due to the pairing of the oxidized adenine in a syn conformation with the cytosine.     

Influence of an 8-oxoadenine lesion on the structural and dynamic features of a 30-mer DNA fragment with and without a mismatch

Purpose : To elucidate the influence of the oxidative lesion 7,8-dihydro-8-oxoadenine (8-oxoA) on the structural and dynamic features of a 30-mer DNA fragment, and to understand if differences occur when C is positioned opposite 8-oxoA instead of T. Materials and methods : Two 30-mer DNA oligomers with or without the 8-oxoA and two complementary oligomers with C or T base opposite the lesion site were synthesized and annealed. Duplexes named AT, A*T, AC and A*C were characterized by means of circular dichroism and UV denaturation measurements. γ -Ray footprinting experiments were performed to give insight into their fine three-dimensional structure. Elastic torsional constants were derived by following the decay of the fluorescence polarization anisotropy (FPA) of the ethidium-DNA complexes measured by multifrequency-phase fluorometry. Results : The introduction of one oxidative lesion in a 30-mer DNA oligomer with and without a mismatch did not cause relevant changes in their overall conformation and slightly modified their elastic properties. Small energetic differences were revealed by thermodynamic analysis in the sample bearing both the oxidative lesion and the mismatch. Minor variations in the cleavage pattern due to the hydroxyl radicals in the A*T sample were observed and present along the entire DNA fragment length. In the A*C sample, by contrast, there was a major modification in the cleavage pattern extending for about 11 bases around the lesion, especially towards the 5'-end. Conclusions : Differences in the fine structure and in the elastic properties between the A*T and A*C samples were observed, while their overall conformation was unchanged. The results are consistent with the hypothesis that the observed local changes of the double helix structure in A*C are due to the pairing of the oxidized adenine in a syn conformation with the cytosine.     

DNA radiolysis in DNA–protein complexes: a stochastic simulation of attack by hydroxyl radicals

Purpose: To propose an improved version of RADACK, a stochastic simulation of radiolytic attack on DNA, that takes into account the reactivity of each amino acid of a specifically bound protein with hydroxyl radicals. To apply it to the natural lactose operator–repressor complex taking advantage of recently reported structures. To compare the obtained probabilities of DNA strand break induction with those calculated with the previous versions and with an experimental pattern of strand break probabilities.

Materials and methods: Models of complexes close to the natural ones, derived from crystallography‐ and NMR‐based structures recently available in the PDB databank, were used. The specific chemical reactivity of each amino acid was introduced in the new version of RADACK (the reactivity model). The probabilities of strand break induction by the irradiation of the complex were calculated with this new version as well as with previous ones.

Results: The patterns of probabilities of strand break induction calculated with the improved version of RADACK were partially different from those obtained with previous versions. The patterns obtained for both, using putative models of natural complexes, were consistent with the experimental results, but some discrepancies were suggestive of slight differences between these structures and the real natural system. The crystallographic structure agreed best with the experimental results.

Conclusions: A new version of RADACK was validated that took into account the reactivity of atoms in both DNA and protein. The putative modelled structures of a natural lactose operator–repressor complex were discussed.     

Hydroxyl radical scavenging by carnosine and Cu(II)-carnosine complexes: a pulse-radiolysis and spectroscopic study

Purpose: To obtain a wider insight into the general properties of carnosine and to provide support to its anti-oxidative role. This property is hypothesized to be linked to various mechanisms including free-radical scavenging and metal chelation (i.e. Cu(II)). Methods: Pulse-radiolysis experiments were performed by a 12MeV electron linear accelerator (LINAC) on carnosine/ copper(II) (2:1) and carnosine aqueous solutions at different pH. Raman spectra of solid samples were obtained by a Bruker IFS 66 spectrometer. Results: As well as for free carnosine, in the presence of copper ions the interaction of carnosine with OH radicals involves the imidazole group of the molecule. The oxidation of copper (II)-carnosine system by OH radicals is related to the pH-dependent structure of the copper(II)-carnosine complex. Raman spectra indicate that at alkaline pH the formation of a dimeric species containing two carnosine molecules complexed to two Cu2+ ions takes place. This structure can address the OH attack more selectively than carnosine itself to different sites of the imidazole ring. The formation of at least two different OH-radical adducts occurs and positions C(2) and C(5) of the imidazole ring are the preferential sites for the OH attack, as the heterocyclic ring is mainly present as its N(1)-protonated tautomeric form. Conclusion: These studies provide further evidence about the formation of carnosine-copper complexes and the predominance of a dimeric structure at slightly basic pH. The chelation of Cu(II) is not detrimental to the scavenging ability of carnosine. Raman spectra are helpful in identifying the structure of the copper(II)-carnosine complexes and in predicting the preferential sites for the OH attack to the carnosine-copper system.     

Background Radiation as the Cause of Fatal Congenital Malformation

Purpose: To investigate the potential antioxidative role of carnosine (beta -alanyl-L-histidine) through its interaction with radiation induced radicals. Materials and methods: Pulse radiolysis experiments were performed by a 12MeV electron linear accelerator (LINAC) on carnosine aqueous solutions at different pHs. The Raman spectra of solid samples were obtained by a Bruker IFS 66 spectrometer. Results: The protonation state of the imidazole ring was observed to affect the site(s) of OH attack on carnosine as well as the decay rates of the resulting adducts. Reasonably, a base catalysed water elimination from the adducts leading to a resonancestabilized radical, which absorbs at lambda 270nm, takes place. Raman spectra in slightly alkaline medium have indicated that carnosine is mainly present as tautomer I and, of consequence, positions C(2) and C(4) of the imidazole ring are the preferential sites for OH attack. Conclusions: These studies have shown that carnosine is a good scavenger of OH radicals giving rise to a quite stable intermediate which should be less reactive towards other biological components than OH itself. Raman data have been helpful in predicting the preferential sites for the OH attack.     

Species identification based on the point mutations of histo-blood group ABO genes by PCR-RFLP and direct sequencing

Using the PCR-restriction fragment length polymorphism (RFLP) and direct sequencing methods, 181 bp DNA fragments at the ABO blood group locus of human and eight different primates were examined. Through PCR amplification and digestion of the product with Hha-1 restriction enzyme, each of the amplified fragments of human, chimpanzee and green monkey was cut into two fragments of 147 and 34 bp, and the corresponding fragment of the other primates was digested into three fragments of 115, 34 and 32 bp. The 181 bp fragments of chimpanzee and green monkey were digested with Mva-1 into three fragments of 82, 58 and 41 bp, and 69, 58 and 54 bp, while the fragments of human and the other primates were separated into two fragments of 123 and 58 bp. Thus, using Hha-1 and Mva-1, human PCR-amplified product of a part of the ABO gene could be discriminated from those of the other primates examined. In addition, by direct sequencing of the 181 bp DNA fragment, two Mva-1 recognition sites and one Hha-1 recognition site were found in the fragments of chimpanzee and green monkey, one Mva-1 site and one Hha-1 site were detected in humans, and one Mva-1 site and two Hha-1 sites in the other primates. These results corresponded well with the data of PCR-RFLP. This method has a good potential for species identification at the DNA level. Received: 27 January 1997 / Received in revised form: 17 March 1997     

DNA ligands as radioprotectors: molecular studies with Hoechst 33342 and Hoechst 33258.

Following earlier reports of radioprotection of cells by Hoechst 33342, we have investigated radioprotection of isolated DNA by the minor groove binders Hoechst 33258 and Hoechst 33342. Analysis of radiation-induced single strand breakage in plasmid DNA (pBR322) showed concentration-dependant protection, up to a dose-modifying factor of 9.3 for 25 microM Hoechst 33258, at which the ligand: bp ratio was 0.67. Since the ligands bind at discrete sites along DNA, sequencing gel analysis was used to investigate the radioprotective effects of the ligands both at and between the ligand-binding sites. These experiments showed that although protection was more pronounced at the binding sites, there was also some reduction in strand-breakage between binding sites. Detailed analysis at a particular site, the EcoR1 site in a 3'-32P-endlabelled 100bp restriction fragment from pBR322, showed that protection was most pronounced at the 'inner T': GAATTC. Irradiation of a synthetic oligodeoxynucleotide containing a single ligand-binding site, and labelled at the 5'-end, gave the expected doublet bands in high resolution gels, corresponding to fragments with 3'-phosphoryl- and 3'-phosphorylglycollate terminii. In the Hoechst 33258-protected sample, the 3'-phosphorylglycollate band was preferentially suppressed within the binding site. These results, together with published crystal structure data for a Hoechst 33258/dodecamer complex, suggest that the site-specific radioprotection may be due to H-atom donation from the benzimidazole NH groups in the ligand to radiation-induced radicals on 4'-deoxyribosyl carbons. In contrast to the experiments with purified DNA, in which the two ligands yielded similar results, Hoechst 33342 was a much more active radioprotector in experiments with intact cells. For 20 microM Hoechst 33342, the dose-modifying factor was 1.7 at 1% survival and 1.3 at 10% survival, whereas the same level of Hoechst 33258 yielded barely detectable protection, perhaps due to a demonstrably lower cellular uptake. Presumably the radioprotection of cells by Hoechst 33342 is due to suppression of DNA strand breakage, and further investigation of the protection mechanism(s) should enable development of improved radioprotectors.