Helix-coil transition in nucleoprotein: renaturation of polylysine-DNA and polylysine-nucleohistone complexes

Thermal denaturation and renaturation of directly mixed and reconstituted polylysine–DNA, directly mixed polylysine–nucleohistone complexes, and NaCl-treated nucleohistones in 2.5 × 10^?4 M EDTA, pH 8.0 have been studied. At the same input ratio of polylysine to DNA, the percent of renaturation of free base pairs in a directly mixed polylysine–DNA complex is higher than that in a reconstituted complex. For a directly mixed complex, the renaturation of free base pairs is proportional to the fraction of DNA bound by polylysine or inversely proportional to the sizes of free DNA loops. A of large amount of renaturation of free base pairs has also been observed for 0.6 M and 1.6 M NaCl-treated nucleohistones. The binding of polylysine to nucleohistone enhances the renaturation of histone-bound base pairs. The percent of renaturation of polylysine–bound base pairs is high and is approximately independent of the extent of binding on DNA by polylysine. This is true in polylysine–DNA complexes prepared either by reconstitution or by directly mixing. It also applies for polylysine–nucleohistone complexes. The model where polylysine-bound base pairs collapse at T_m′ with two complementary strands still bound by polylysine is favored over the model where polylysine is dissociated from DNA during melting. The low renaturation of histone-bound base pairs in nucleo-histone indicates that either histones do not hold two complementary strands of DNA tightly or that histones are fully or partially dissociated from DNA when the nucleo-histone is fully denatured.     

Protein-DNA crosslinking in reconstituted nucleohistone, nuclei and whole cells by picosecond UV laser irradiation.

A picosecond UV laser was used to cross-link proteins to DNA in nuclei, whole cells and reconstituted nucleohistone. Irradiation of the nucleohistone resulted in crosslinking 15-20% of bound histones to DNA in a very short time (one or several picosecond pulses), the efficiency of crosslinking to single stranded DNA being higher than to double stranded DNA. All histones as well as high mobility group 1 proteins were identified in the covalently linked protein-DNA complexes upon irradiation of isolated nuclei and whole cells. A method is suggested for isolation of crosslinked material from cells and nuclei in amounts sufficient for further analysis. Experiments with reconstituted nucleohistones showed that upon irradiation at a constant dose the efficiency of crosslinking depended on the intensity of the light, thus suggesting a two-quantum process is involved in the reaction.     

Circular dichroism of native and reconstituted nucleohistones

Circular dichroism (CD) spectra of histone, DNA and native, partial and reconstituted nucleohistone complexes in 0.01 M Tris buffer in which nucleohistone complexes appear to be intact and in 2 M NaCl, in which complexes are mostly dissociated, have been compared. The CD spectra in the longwave region, where the protein contribution is negigible, reveal differences in the spectra for DNA in nucleohistone complexes as compared with that of free DNA, the difference being roughly proportional to the protein DNA ratio. An analysis of the shape of the first positive CD maximum suggests that we are probably dealing here with slight conformational changes on DNA. Minor differences between the behavior of native and reconstituted complexes may be due to the effect of different conformational states of the protein component.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

The dissociation of histone from deoxyribonucleohistone by γ-irradiation

1. Experiments were carried out to determine the extent of dissociation of histone from deoxyribonucleohistone as a result of irradiation with γ-rays from ⁶⁰Co. 2. The bulk of the nucleohistone was removed from the irradiated solutions either by sedimentation or by precipitation with dilute sodium chloride solution. The supernatants were then analysed for DNA and histone. 3. The ratios of histone to DNA in these supernatants were less than for the original nucleohistone. This indicated that histone was dissociated by the irradiation, and then aggregated either with itself or with other nucleohistone molecules, and so was removed with the bulk of the nucleohistone during sedimentation or precipitation.     

Localization of chromatin proteins within DNA grooves by methylation of chromatin with dimethyl sulphate

The use of the comparative modification with ³H-dimethyl sulphate (DMS) of free DNA and DNA in different complexes is proposed to evaluate the shielding of the minor and major grooves of the DNA double helix and to determine the presence of single-stranded DNA in the complexes.Glucosyl groups in DNA of T6 phage protect, as expected, the major groove, and actinomycin d in its complex with DNA shields the minor groove against methylation with DMS.The data obtained suggest that histones and protamine in reconstituted nucleohistone and nucleoprotamine are allocated within partly the major groove leaving the minor groove open, while polylysine does not seem to be buried within either of the grooves, and cations of cetyltrimethylammonium lie within the minor groove of DNA.     

Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

Studies on interaction between histone V (f2c) and deoxyribonucleic acids.

Histone V (2fc) from chick erythroctes was used in the study of its interaction with DNA from various sources. Complexes between this histone and DNA were formed using the procedure of continuous NaCl gradient dialysis in urea. Two physical methods, namely thermal denaturation and circular dichroism (CD), were used as analytical tools. Thermal denaturation of nucleohistone V with chick or calf thymus DNA shows three melting bands: band I at 45-50 degrees corresponds to free base pairs; band II at 75-79 degrees, and band III at 90-93 degrees correspond to histone-bound base pairs. In histone-bound regions, there are 1.5 amino acid residues/nucleotide in nucleohistone V. In contrast, a value between 2.9 and 3.3 was determined for nucleohistone I (fl) (H. J. Li (1973), Biopolymers 12, 287). Similar melting properties have been observed for histone V complexed with bacterial DNA from Micrococcus luteus. Histone V binding to DNA induces a slight transition from a B-type CD spectrum to a C-type spectrum. Trypsin treatment of nucleohistone V reduces melting band III much more effectively than band II. Such a treatment also restores DNA to B conformation in the free state. Reduction of the melting bands of nucleohistone V by polylysine binding follows the order of I greater than II greater than III, accompanied by the increase of a new band at 100 degrees. When two bacterial DNAs of varied A + T (adenine + thymine) content simultaneously compete for the binding of histone V, the more (A " T)-rich DNA is selectively favored. Under experimental conditions described here, Clostridium perfringens DNA with 69% A + T is bound by histone V in preference to chicken DNA with 56% A + T although the latter has natural sequences for histone V binding.     

Studies on radiation-sensitive mutants of E. coli. 3. Participation of the rec system in induction of mutation by ultraviolet irradiation

Summary The bacterial recA gene participates in the induction by UV irradiation of the clear mutation of phage and the Lac^- mutation of bacteria. The necessary function is induced by irradiation of Rec⁺ bacteria and acts upon DNA irradiated with UV light.     

Inducible reactivation of UV-irradiated cholera phage e5 in Vibrio cholerae MAK757

Summary The survival of UV-irradiated cholera phage e5 was found to increase when the host cells, Vibrio cholerae MAK757, were exposed to a low dose of UV irradiation before phage infection (Weigle reactivation), indicating the existence of a UV-inducible DNA repair pathway (SOS repair) in V. cholerae MAK757. The induction signal generated by UV irradiation was transient in nature and lasted about 20–30 min at 37°C. Maximal weigle reactivation of the phage was obtained when the host cells were irradiated with a UV dose of 16 J/m². V. cholerae MAK757 was also found to possess efficient photoreactivation and host cell reactivation of UV-damaged DNA in phage e5.     

Distribution of histone F1 on calf thymus nucleohistone DNA.

A method of large-scale preparation of the histone F1-DNA complex by removing all other proteins from calf thymus nucleohistone was established. This involved gel filtration of nucleohistone through a column containing a band of sodium dodecyl sulfate. The F1-DNA complex obtained had the original amount of F1 and no other. The F1-DNA complex exhibited distinct two-step melting on thermal denaturation. The first step was apparently attributable to naked DNA regions and the second step, about 30 deg. C higher than the first step, to the regions covered with F1. Buoyant density experiments with the complex after fixation with formalin revealed that F1 was distributed fairly evenly over DNA fragments of an average molecular weight of about 4 × 10⁶. Electron microscopic examination of the complex after various degrees of denaturation with formalin indicated that the longest stretch of unbound DNA was about 0·3 μm.     

Effect of ultraviolet radiation on cell division and microtubule organization inPetunia hybrida protoplasts

Summary Mesophyll protoplasts isolated fromPetunia hybrida were subjected to UV radiation (280–360 nm) in an attempt to assess whether (a) UV radiation has an effect on cortical microtubule organization, (b) UV radiation affects the progression of protoplasts through the cell cycle, and (c) there is a connection between the effect of UV radiation on cell division and the polymerization state of the microtubules. The proto plasts were irradiated with the following UV doses: 4, 8, 12, and 24mmol photons/m², 30 min after isolation. Cell cycle analysis and immuno-localization of microtubules were carried out 0, 24, 48, and 72 h after irradiation. The length of cortical microtubules was determined after irradiation and in corresponding controls. We found that UV radiation induced breaks in cortical microtubules resulting in shorter fragments with increasing dose. Also, the protoplasts were delayed in their progression through the cell cycle, with G1 and G2 phases being affected as well as the S phase. The commencement of DNA synthesis in the irradiated protoplasts followed the re-establishment of a microtubule network. At 48 h after irradiation the protoplasts in all treatments, except for the 24 mmol/m², had cortical microtubules of similar length, and at 72 h after irradiation only the protoplasts that had received 24 mmol photons/m² had not started dividing.Abbreviations BSA bovine serum albumin - DMSO dimethyl sulfoxide - FDA fluorescein diacetate - MT microtubules - MTSB microtubule stabilizing buffer - PAR photosynthetically active radiation (400–700 nm) - PBS phosphate buffered saline - UV ultraviolet     

The effect of low ionic strength on the radiation chemistry and physical properties of calf thymus DNA

Studies of ultraviolet and circular dichroism spectra of aqueous solutions of calf thymus (CT) DNA confirm the tendency of DNA to change conformation at low ionic strength. The qualitative shape and transition width of 260 nm melting curves below 1 mM NaCl differed significantly from those previously published for DNA solutions containing 1 mM NaCl and above. Neutral aqueous solutions of CT DNA at low ionic strengths (0.1 mM-10 mM NaCl) were irradiated with low doses of gamma-rays. The melting temperature, Tm, of irradiated DNA samples increased below 1 mM NaCl suggesting interstrand crosslinking of the denatured DNA or formation of regions of more thermally stable DNA conformation. The magnitudes of these radiation responses were found to be a function of the time elapsed between salt concentration changes and irradiation as well as time after irradiation. These results are consistent with the hypothesis that the purine and pyrimidine base chromophores in double stranded DNA are sheltered from radical attack by the sugar phosphate backbone. Low dose radiation studies (0.8-8.0 Gy) of CT DNA in 1 mM NaCl and below showed a split dose and dose rate dependence for the sample melting curves.     

Effect of gamma radiation and accelerated electron beam on stable paramagnetic centers induction in bone mineral: influence of dose,irradiation temperature and bone defatting

Ionizing radiation has been found to induce stable defects in the crystalline lattice of bone mineral hydroxyapatite, defined as CO₂ ^? radical ions possessing spins. The purpose of our study was to evaluate CO₂ ^? radical ions induced in non-defatted or defatted human compact bone by gamma radiation (G) and accelerated electron beam (EB), applied with two doses at different temperatures. Moreover, the potential effect of free radical ion formation on mechanical parameters of compact bone, tested under compression in the previous studies, was evaluated. Bone rings from femoral shafts of six male donors (age 51 ± 3 years) were collected and assigned to sixteen experimental groups according to different processing methods (non-defatted or defatted), G and EB irradiation dose (25 or 35 kGy), and irradiation temperature [ambient temperature (AT) or dry ice (DI)]. Untreated group served as control. Following grinding under LN₂ and lyophilization, CO₂ ^? radical ions in bone powder were measured by electron paramagnetic resonance spectrometry. We have found that irradiation of bone with G and EB induces formation of enormous amounts of CO₂ ^? radical ions, absent from native tissue. Free radical ion formation was dose-dependent when irradiation was performed at AT, and significantly lower in EB as compared to G-irradiated groups. In contrast, no marked effect of dose was observed when deep-frozen (DI) bone samples were irradiated with G or EB, and free radical ion numbers seemed to be slightly higher in EB-irradiated groups. Irradiation at AT induced much higher quantities of CO₂ ^? radical ions then on DI. That effect was more pronounced in G-irradiated bone specimens, probably due to longer exposure time. Similarly, bone defatting protective effect on free radical ion formation was found only in groups irradiated for several hours with gamma radiation at ambient temperature. Ambient irradiation temperature together with exposure time seem to be key parameters promoting CO₂ ^? radical ion formation in bone mineral and may mask the opposite effect of defatting and the possible effect of irradiation type. Significant weak negative correlations between CO₂ ^? radical ion number and some mechanical properties of compact bone rings (Young’s modulus and ultimate stress) were found.     

Dependence of the U.V. Survival of transforming DNA on the amount of DNA uptake per cell

Summary UV irradiation of transforming DNA from Haemophilus influenzae, carrying a streptomycin resistance marker (Sr), results in decreased transforming activity. At high DNA concentration the marker survival is lower than it is at low concentration. The transition from high to low survival occurs at concentrations ranging from 2.5×10^-3 to 2.5×10^-2 g/ml; in this range the probability that transformed cells take up DNA fragments in addition to the marked one increases rapidly. A similar effect of DNA concentration on the percentage of transformants is observed for a mixture of unirradiated and irradiated DNA, where virtually all of the transformants originate from the unirradiated component. This eliminates the possible explanation that the concentration dependence of UV survival of a marker reflects increasing competition for a cellular repair system.It is concluded that the lower marker survival obtained at high DNA concentration involves lethality due to UV lesions present in the additional irradiated DNA taken up by the cell. Thus the steeper marker survival curve is due to the increasing UV dose which the additional DNa necessarily receives when a marker survival curve is being established. Intergration of UV lesions rendering a chromosomal DNA strand inviable is suggested by a slight delay in cell multiplication after uptake of irradiated and — to a lesser extent — unirradiated DNA. Acriflavine at a concentration of 0.5g/ml enhances the effect of DNA concentration on marker survival. Similarly the number of transformants obtained with unirradiated DNA in the presence of acriflavine is more strongly decreased at high than at low DNA concentration. It is suggested that each event of DNA integration involves a small change for lethality, which is enhanced if the DNA carries UV lesions or if acriflavine is present.Dedicated to Professor H. Nachtscheim on the occasion of his 80th birthday.     

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

Summary In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5¹, 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that base-pairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.     

Effect of glutathione on UV induction of prophage lambda

The effect of glutathione (GSH) on the ultraviolet (UV) induction of lambda prophage was investigated in lysogenic Escherichia coli. The data showed that extracellular GSH could inhibit the UV induction of lambda prophage. The inhibitory rates were concentration dependent, and the maximal rate obtained was 94% with 3.0 M GSH. The effect was also measured in three different lambda lysogens: a wild-type strain (wt), an isogenic GSH-deficient strain, and an isogenic strain producing increased amounts of GSH. The result showed that when subjected to UV irradiation (254 nm, 60 J m⁻²), GSH-deficient strain was approximately fivefold more sensitive to be lysed than wt, whereas the strain with higher intracellular GSH levels was only 28% susceptible to be lysed. With electron spin resonance and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated lysogenic cells and the intensity of signals was influenced by GSH levels. These results indicate that GSH can significantly inhibit the UV induction of lambda prophage, and that this effect is correlated to its capacity to scavenge free radicals generated after UV irradiation.     

The effect of cold after whole-body irradiation of the mouse

Mice were placed in a cold environment (4 °C) directly after whole-body irradiation. Those irradiated with a lethal dose showed higher lethality than mice irradiated with the same dose but placed in room temperature. The response was also altered after irradiation with a sublethal dose. At various periods after irradiation mice were injected with¹²⁵IUdR, the tissue uptake of which is an index of DNA synthesis. The result showed that cold treatment after irradiation caused slower cell renewal in the spleen and bone marrow, but that the thymus was only marginally affected. Furthermore, the concentrations of erythrocytes in the peripheral blood reached a lower level in the cold-treated group. Finally, the levels of the thyroid hormones T3 and T4 in the blood were measured and it was found that the T3/T4 ratio was higher in the cold-treated mice. It is suggested that during prolonged exposure to cold after irradiation the cell recovery in the haemopoietic system is exposed to hormonal action that induces significant alterations in the postirradiation cell kinetics.The animal experiments described herein were approved by the Regional Research Ethical Committee according to the Swedish National Laws SFS 1988:539 and LSFS 1989:41     

Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant.

Summary Preincubation at 42^o, before infection at permissive temperature by phage , of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage. Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair. This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating DNA, probably due to induction of diffusible products.     

Efficacy of UV Irradiation in Inactivating Cryptosporidiumparvum Oocysts

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log₁₀ reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm² at 20°C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm² for a 2-log₁₀ reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm². Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10°C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log₁₀ reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.