Distribution of histones in alkali-denatured chromatin studied by isopycnic centrifugation in alkaline metrizamide density gradients.

Three types of density gradients - neutral metrizamide, alkaline NaOH-metrizamide and alkaline triethanolamine-metrizamide - were used for studying the distribution of histones between the two DNA strands in alkali-denatured chromatin. It was found possible to avoid both protein redistribution and dissociation by using triethanolamine-metrizamide density gradients at pH 10.5. Under these conditions an alkali-denatured mixture of DNA and chromatin was well separated into the original DNA and DNP. When native or sonicated chromatin was denatured at pH 12.2 and centrifuged in a triethanolamine-metrizamide density gradient at pH 10.5 no peak of free DNA appeared. These results show that both DNA strands remain associated with histone molecules upon alkaline denaturation of chromatin.     

Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.     

DNA polymerase eta reduces the gamma-H2AX response to psoralen interstrand crosslinks in human cells

DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase eta are sensitive to psoralen photoadducts created under conditions favoring crosslink formation, suggesting a role for translesion synthesis in crosslink repair. Because crosslinks can lead to double-strand breaks, we monitored phosphorylated H2AX (gamma-H2AX), which is typically generated near double-strand breaks but also in response to single-stranded DNA, following psoralen photoadduct formation in XP-V fibroblasts to assess whether polymerase eta is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced gamma-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase eta in XP-V cells normalized the gamma-H2AX response. In response to psoralen crosslinking, gamma-H2AX as well as 53BP1 formed coincident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced gamma-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase eta is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase eta are involved in processing crosslinks and avoiding gamma-H2AX associated with double-strand breaks and single-stranded DNA in human cells.     

Electron microscopic identification of supercoiled regions in complex DNA structures

When intracellular lambda replicative intermediates (theta structures) are intercalated with psoralen and then irradiated with long wavelength ultraviolet light (u.v.), interstrand crosslinks are produced. After purification and denaturation of these theta structures, a global difference in denaturation can be observed by electron microscopy; parental sections are essentially native whereas daughter segments are highly denatured. This difference can be explained if parental sections are covalently continuous (and therefore able to supercoil) and daughter segments are not. Due to the higher thermal stability of supercoiled DNA, parental DNA will remain native while daughter sections will denature. Because these structures are crosslinked, the thermal treatment does not lead to dissociation of the highly denatured daughter strands. Experiments with simple negatively supercoiled plasmid circles support the above conclusions. When circles are crosslinked with psoralen-u.v. and then denatured, they remain native because of the higher thermal stability of covalently closed structures. If the circles are linearized before heating but after the psoralen-u.v. treatment, the thermal stability effect is eliminated and the molecules become highly denatured. In this case, however, the crosslinking density is found to be higher than in samples linearized before psoralen-u.v. treatment. This, therefore, shows that crosslinking density also reflects the superhelical state of the molecule at the time of psoralen-u.v. treatment. Two different properties can be used to discriminate between supercoiled and covalently discontinuous domains in complex DNA structures. First, supercoiled regions remain native while covalently discontinuous segments denature following a thermal treatment. This effect requires that covalent continuity exists up to and during the heating treatment. Second, because negative superhelicity enhances psoralen intercalation, crosslinking density is higher in these regions. Even if supercoiled domains are destroyed after the psoralen-u.v. treatment, the imprint of superhelicity is retained and can be recognized as a higher than normal crosslinking density.     

Psoralen-DNA crosslink repair in human lymphocytes: Comparison of alkaline elution with electron microscopy

Much interest has surrounded the question of the removal of psoralen interstrand crosslinks in DNA of eukaryotic organisms. A commonly employed method for the study of psoralen repair is alkaline elution. In this study we have used alkaline elution to assess psoralen crosslink repair in human lymphocytes. The lymphocytes were treated with 8-methoxypsoralen or 4,5′,8-trimethylpsoralen and allowed to repair for different periods of time. Analysis by alkaline elution showed elution patterns compatible with crosslink removal. When the crosslink removal under comparable conditions was studied by the use of electron microscopy under totally denaturing conditions, no repair of the crosslinks could be detected.     

Natural Occurrence of Cross-Linked Vaccinia Virus Deoxyribonucleic Acid

The molecular weight of native vaccinia deoxyribonucleic acid (DNA) is 1 to 1.17 times that of native T4 DNA. Sedimentation of denatured vaccinia DNA through alkaline sucrose gradients yields an apparent molecular weight greater than twice that of denatured T4 DNA, implying that the complementary strands of vaccinia DNA do not separate upon denaturation. When alkali-denatured vaccinia DNA is neutralized, it has the physical chemical properties of native DNA when tested by sedimentation through neutral sucrose gradients, banding in CsCl, and by hydroxylapatite chromatography. We conclude that almost all mature vaccinia DNA molecules contain a small number of naturally occurring cross-links.     

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.     

Repair of 8-methoxypsoralen monoadducts in mouse lymphoma cells

Studies of the repair of DNA lesions at biologically important doses is extremely difficult for most mutagens. With 8-methoxypsoralen (8-MOP) plus longwave ultraviolet light (UVA) as the lesion-inducing agent, however, it is easy to manipulate the relative frequency of different DNA adducts by means of a special experimental protocol (the tap-and-test protocol) and this can be used to measure repair of DNA adducts. Three classes of photoadducts are produced by 8-MOP plus UVA treatment: 3,4-cyclobutane monoadducts, 4',5'-cyclobutane monoadducts, and 8-MOP-DNA interstrand crosslinks. A monoadduct is formed when a photoactivated 8-MOP molecule reacts with a pyrimidine base. An 8-MOP-DNA interstrand crosslink is formed when an existing monoadduct is photoactivated to react with another pyrimidine base on the opposite DNA strand. Thus monoadducts are formed by absorption of one photon of light and crosslinks by absorption of two. In the tap-and-test experiments, cells were exposed to UVA in the presence of 8-MOP and then re-exposed to UVA in the absence of free 8-MOP so that only crosslinks can be produced by the second UVA treatment. By means of this technique we have previously shown that DNA crosslinks are much more effective than monoadducts at producing chromosomal damage (sister-chromatid exchanges and micronuclei) but not mutations (Liu-Lee et al., 1984). If L5178Y mouse lymphoma cells were able to remove monoadducts, incubation prior to the second UVA treatment should lead to decreases in the effect of re-irradiation, because fewer monoadducts would be available for crosslink formation. In this way, we have found that psoralen monoadducts are repaired in these cells and that about 70% of those capable of crosslink formation are removed or otherwise made unavailable for crosslink formation in 6 h.     

In vitro crosslinking of DNA by 8-methoxypsoralen visualized by electron microscopy

By electron microscopic visualisation of totally denatured DNA, we have detected photochemically induced 8-methoxypsoralen crosslinks in vitro after irradiation at 360 nm. The amount of crosslinks was expressed as the percentage of DNA length which was kept in double-stranded appearance by closely situated crosslinks. This percentage correlated well with irradiation time, irradiation intensity, and the concentration of 8-methoxypsoralen. These parameters have also been correlated with the mean size and the size distribution of non-crosslinked regions of DNA, so called bubbles. For a comparison with another psoralen type, we have carried out a similar set of experiments using 4,5,8-trimethylpsoralen.     

Mechanical properties of a reversible, DNA-crosslinked polyacrylamide hydrogel

Mechanical properties of a polyacrylamide gel with reversible DNA crosslinks are presented. In this system, three DNA strands replace traditional chemical crosslinkers. In contrast to thermoset chemically crosslinked polyacrylamide, the new hydrogel is thermoreversible; crosslink dissociation without the addition of heat is also feasible by introducing a specific removal DNA strand. This hydrogel is characterized by a critical crosslink concentration at which gelation occurs. Below the critical point, a characteristic temperature exists at which a transition in viscosity is observed. Both temperature-dependent viscosity and elastic modulus of the material are functions of crosslink density.     

Sensitivity of mitomycin C and nitrogen mustard crosslinks to extreme alkaline conditions

DNA-DNA crosslinks in cells treated with mitomycin C, nitrogen mustard, or decarbamoyl mitomycin C were measured in alkaline isopycnic gradients as a function of pH. Crosslinks from cells treated with mitomycin C and nitrogen mustard, which react with DNA purines, could be detected at pH 12.5 but not at pH 14. No crosslinks from cells treated with decarbamoyl mitomycin C were detected at either pH. Previous studies with cells exposed to psoralen derivatives plus 360 nm light, which produce DNA-DNA crosslinks with pyrimidines, demonstrated stable crosslinks at pH 14. These studies indicate that DNA-DNA crosslinks involving DNA purines are much less stable at high pH than those involving pyrimidines, and that methods involving exposure to extreme alkaline conditions may give inaccurate information for some agents.     

Failure of signaling through a chimeric class I-immunoglobulin molecule expressed on the surface of transfected B lymphoma cells and cells of transgenic mice.

To test the possibility that the crosslinkage of molecules expressing a transmembrane region derived from the membrane form of the mu immunoglobulin heavy chain would be sufficient for signal transduction in B cells, a chimeric gene (Kk-mu) consisting of extracellular exons of the class I gene H-2Kk and the transmembrane and cytosolic exons of the mu constant region gene was introduced into WEHI-231 B lymphoma cells and into mouse blastocysts. A protein consistent with the predicted product of the Kk-mu gene was expressed in a transfected cell clone (S18) and in transgenic mice. Crosslinkage of Kk-mu protein with soluble, Sepharose-bound, or dextran-conjugated anti-H-2Kk antibodies failed to induce the accumulation of inositol phosphates or to elevate intracellular calcium concentrations in either S18 cells or B lymphocytes from transgenic mice. Furthermore, crosslinkage of Kk-mu did not inhibit growth of S18 cells or stimulate DNA synthesis by transgenic B cells, in the presence or absence of interleukin-4. The failure of crosslinkage of Kk-mu to initiate detectable intracellular biochemical change or to effect cellular growth suggests that simple crosslinkage of molecules expressing the mu transmembrane region is not sufficient to transduce signals in B cells.     

Determination of hepatitis B virus DNA in serum by molecular hybridization

Hepatitis B virus (HBV) DNA was detected by direct spotting of alkali-denatured serum on a nitrocellulose filter and molecular hybridization with cloned HBV DNA as the probe. Measurement of the autoradiographic signals as the intensity of hybridization allowed the quantitation of HBV DNA content in serum specimens in reference to cloned HBV DNA. Direct spotting of denatured serum was approximately three times as sensitive as the conventional method in which proteinase-treated serum was extracted with phenol-chloroform. The intensity of hybridization with 25 specimens of HB virion concentrates correlated well with DNA polymerase activity (r = 0.89, P less than 0.01).     

Crosslinking studies on the organization of the 16 S ribosomal RNA within the 30 S Escherichia coli ribosomal subunit.

The location and frequency of RNA crosslinks induced by photoreaction of hydroxymethyltrimethylpsoralen with 30 S Escherichia coli ribosomal subunits have been determined by electron microscopy. At least seven distinct crosslinks between regions distant in the 16 S rRNA primary structure are seen in the inactive conformation of the 30 S particle. All correspond to crosslinked features seen when the free 16 S rRNA is treated with hydroxymethyltrimethylpsoralen. The most frequently observed crosslink occurs between residues near one end of the molecule and residues about 600 nucleotides away to generate a loop of 570 bases. The size and orientation of this feature indicate it corresponds to the crosslinked feature located at the 3′ end of free 16 S rRNA.When active 30 S particles are crosslinked in 5 mm-Mg²⁺, six of the seven features seen in the inactive 30 S particle can still be detected. However, the frequency of several of the features, and particularly the 570-base loop feature, is dramatically decreased. This suggests that the long-range contacts that lead to these crosslinks are either absent or inaccessible in the active conformation. Crosslinking results in some loss of functional activities of the 30 S particle. This is consistent with the notion that the presence of the crosslink that generates the 570-base loop traps the subunit in an inactive form, which cannot associate with 50 S particles.The arrangement of the interacting regions crosslinked by hydroxymethyltrimethylpsoralen suggests that the RNA may be organized into three general domains. A striking feature of the Crosslinking pattern is that three of the seven products involve regions near the 3′ end of the 16 S rRNA. These serve to tie together large sections of rRNA. Thus structural changes at the 3′ end could, in principle, be felt through the entire 30 S particle.     

Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.     

The effect of trimethylpsoralen--crosslinks on entry of donor DNA in transformation and transfection of Bacillus subtilis

Summary Transforming chromosomal DNA, irradiated with long-wave UV light in the presence of 4,5,8-trimethylpsoralen (TMP) binds to competent B. subtilis cells as effectively as non-treated DNA, but its transforming activity is strongly reduced.Uptake studies show that the entry of transforming DNA, after some stimulation by short periods of irradiation in the presence of TMP, decreases proportionally with the dose of irradiation. Crosslinking was quantitated by electron microscopy. Since the number of crosslinks increases proportionally with the dose of irradiation, it is suggested that entry of donor DNA is prevented by crosslinks. The inhibition of entry of DNA is paralleled both by decreased breakdown of crosslinked DNA interacting with competent cells, and decreased breakdown by nuclease activity liberated during protoplasting of competent cultures. These data support the model of Lacks et al. (1976) which postulates that a membrane-bound deoxyribonuclease is engaged in the entry of donor DNA into the competent cell.The transforming activity of the chloramphenicol-resistance carrying plasmid pC194, originally obtained from Staphylococcus aureus, is also destroyed by TMP crosslinks. Contrary to chromosomal DNA, its association with the cells is stimulated by longwave UV irradiation in the presence of TMP, but experiments are presented suggesting that the DNA is still vulnerable to the action of exogenous pancreatic deoxyribonuclease.Transfecting SPP1 DNA is also inactivated by TMP crosslinks. Marker rescue of transfecting DNA containing crosslinks occurs; the extent of rescue of one marker is considerably in excess of that of linked markers.