Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation.

DNA interstrand cross-links are formed by chemotherapy drugs as well as by products of normal oxidative metabolism. Despite their importance, the pathways of cross-link metabolism are poorly understood. Laser confocal microscopy has become a powerful tool for studying the repair of DNA lesions that can be detected by immunofluorescent reagents. In order to apply this approach to cross-link repair, we have synthesized conjugates of 4,5',8-trimethylpsoralen (TMP) and easily detected compounds such as Lissamine rhodamine B sulfonyl chloride (LRB-SC), biotin, and digoxigenin. These conjugates are activated by UVA, and we have analyzed the intracellular localization of DNA damage and DNA reactivity by confocal and immunofluorescence microscopy. The LRB-SC-TMP conjugate 2 appeared mainly in the mitochondria, while the biotin-TMP conjugate 4 preferentially localized in the cytoplasm. Adducts formed by UVA and digoxigenin conjugates of TMP 7a and 4,5'-dimethylangelicin (DMA) 7b, which forms only monoadducts, were largely localized to the nucleus. Exposure of cells incubated with 7a and 7b to a 364 nm UV laser directed toward defined nuclear regions of interest resulted in localized adduct formation which could be visualized by immunofluorescence. Repair-proficient cells were able to remove the photoadducts, while repair-deficient cells were unable to repair the damage. The results indicated that the digoxigenin-TMP conjugate 7a and digoxigenin-DMA conjugate 7b can be used for studying the repair of laser localized DNA monoadducts and cross-links.

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA.

Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so, and likely modulates their repair by the NER machinery. The abundance of HMGB1 suggests that it may play an important role in determining the sensitivity of cells to DNA damage under physiological, experimental, and therapeutic conditions.

Amino acids in SRS1 and SRS6 are critical for furanocoumarin metabolism by CYP6B1v1, a cytochrome P450 monooxygenase.

CYP6B1v1 is the principal cytochrome P450 monooxygenase (P450) that detoxifies dietary furanocoumarins in the guts of Papilio polyxenes, the black swallowtail caterpillar. Sequence alignments and structure comparisons of CYP6B1v1 with the mouse CYP2A5 and bacterial CYP102 proteins, which are also capable of metabolizing the linear furanocoumarin xanthotoxin (8-methoxypsoralen), suggested that Phe116, His117, Val368 and Phe484 might be active site residues. In a homology model developed for CYP6B1v1, the side chains of Phe116 and His117 located in the B'-C loop of SRS1 are predicted to be positioned above the haem plane, while the side chain of Phe484 located in SRS6 is predicted near the entrance of the catalytic pocket. Site-directed mutagenesis of residues Phe116, His117 and Phe484 indicated that these residues represent several of those that determine this protein's stability and substrate specificity. Whereas all aromatic mutants of Phe116 and Phe484 generated CO-difference spectra with maxima at 450 nm indicative of correctly configured monooxygenases, aromatic mutants of Phe116 exhibited reduced reactivities toward some furanocoumarins and aromatic mutants of Phe484 eliminated all reactivities toward furanocoumarins. All single and double aliphatic mutants of Phe116, His117 and Phe484 and aromatic mutants of His117 generated carbon monoxide (CO) difference spectra with maxima at 420 nm (P420) indicative of incorrectly configured monooxygenases. These studies define residues Phe116, His117 and Phe484 as determinants of this insect P450's catalytic site integrity and residues Phe116 and Phe484 as determinants of its substrate specificity. Conservation of Phe116 and His117 in an array of lepidopteran CYP6B proteins implies that these amino acids serve a similar function in other monooxygenases of the insect CYP6B subfamily.

The effects of singlet oxygen produced by photodynamic action on the mitochondrial permeability transition differ in accordance with the localization of the sensitizer.

We have examined whether the effects of singlet oxygen (1O2) produced by photodynamic action on the mitochondrial permeability transition (PT) can be modulated by the localization of photosensitizers in irradiated mitochondria. We have previously shown that oxidation due to 1O2 photogenerated in hematoporphyrin (HP)-loaded mitochondria can prevent opening of the PT pores, likely after degradation of some critical histidines (Salet et al, 1997, J. Biol. Chem. 272, 21938-21943). Equally, in the present study we have irradiated mitochondria in the presence of a structurally different photosensitizer producing 1O2, namely 4,5',8-trimethylpsoralen (TMP). Fluorescence studies show that TMP binds to protein sites which differ from those of HP. In sharp contrast with HP, TMP-driven photodynamic action triggers per se pore opening. Interestingly, this inducing effect is inhibited when TMP-treated mitochondria are irradiated after addition of mersalyl, a specific reagent protecting thiol groups of the inner mitochondrial membrane that are oriented toward the external hydrophilic phase. This fact suggests that 1O2-mediated thiol oxidation is responsible for TMP-photoinduced pore opening. Taken together, these findings suggest that 1O2 can activate or inactivate a cellular function such as mitochondrial PT depending on the site where it is produced in the mitochondrial membrane.

Mapping psoralen cross-links at the nucleotide level in mammalian cells: suppression of cross-linking at transcription factor- or nucleosome-binding sites.

We have developed a new genomic sequencing method for detecting, with resolution at the nucleotide level, the interstrand DNA cross-links induced by 4,5',8-trimethylpsoralen along single-copy genes in mammalian cells. The cross-links (diadducts) initially formed are converted into monoadducts by alkali reversal prior to the use of terminal transferase-dependent PCR (TD-PCR). After alkali reversal, but not before, the DNA strands can be separated and used as templates for gene-specific primer extension, which is the first step in the TD-PCR procedure. The converted psoralen adducts block primer extension, and the prematurely terminated single-stranded products are then amplified by TD-PCR and visualized on a sequencing gel. Adducts formed by angelicin, a psoralen derivative that forms only monoadducts, were also investigated by use of TD-PCR. Comparison of the adduct distribution patterns of in vivo-treated DNA with those of in vitro-treated DNA revealed that the binding of transcription factors inhibited both psoralen cross-linking and angelicin monoadduct formation in the c-JUN and c-FOS promoters in living human cells. Adduct formation was also inhibited in the region of a putative positioned nucleosome in the c-FOS promoter. These methods should be of general use for study of in vivo protein-DNA interactions and DNA repair.

The crystal structures of psoralen cross-linked DNAs: drug-dependent formation of Holliday junctions.

The single-crystal structures are presented for two DNA sequences with the thymine bases covalently cross-linked across the complementary strands by 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). The HMT-adduct of d(CCGCTAGCGG) forms a psoralen-induced Holliday junction, showing for the first time the effect of this important class of chemotheraputics on the structure of the recombination intermediate. In contrast, HMT-d(CCGGTACCGG) forms a sequence-dependent junction. In both structures, the DNA duplex is highly distorted at the thymine base linked to the six-member pyrone ring of the drug. The psoralen cross-link defines the intramolecular interactions of the drug-induced junction, while the sequence-dependent structure is nearly identical to the native Holliday junction of d(CCGGTACCGG) alone. The two structures contrast the effects of drug- and sequence-dependent interactions on the structure of a Holliday junction, suggesting a role for psoralen in the mechanism to initiate repair of psoralen-lesions in mammalian DNA.

Fanconi anemia, complementation group A, cells are defective in ability to produce incisions at sites of psoralen interstrand cross-links.

The hypersensitivity of Fanconi anemia, complementation group A, (FA-A) cells to agents which produce DNA interstrand cross-links correlates with a defect in their ability to repair this type of damage. In order to more clearly elucidate this repair defect, chromatin-associated protein extracts from FA-A cells were examined for ability to endonucleolytically produce incisions in DNA at sites of interstrand cross-links. A defined 140 bp DNA substrate was constructed with a single site-specific monoadduct or interstrand cross-link produced by 4,5',8-trimethylpsoralen (TMP) plus long wavelength (UVA) light. Our results show that FA-A cells are defective in ability to produce dual incisions in DNA at sites of interstrand cross-links. Specifically, there is defective incision on the 3'- and 5'-sides of both the furan and pyrone sides of the cross-link. This defect is corrected in FA-A cells transduced with a retroviral vector expressing FANCA cDNA. At the site of a TMP monoadduct, FA-A cells can introduce incisions on both the 3'- and 5'-sides of the furan side monoadduct, but are defective in ability to produce these incisions on the pyrone side monoadduct. These studies also indicate that XPF is involved in production of the 5' incision by the normal extracts on these substrates. These results correlate with our previous work, which showed that FA-A cells are mainly defective in ability to repair psoralen interstrand cross-links with a lesser defect in ability to repair psoralen monoadducts. This defect in endonucleolytic incision at sites of TMP interstrand cross-links could be related to reduced levels of non-erythroid alpha spectrin (alphaSpIISigma*) in the extracts from FA-A cells. alphaSpIISigma* could act as a scaffold to align proteins involved in cross-link repair and enhance their interactions; a deficiency in alphaSpIISigma* could thus lead to reduced efficiency of repair and the decreased levels of incisions we observe at sites of interstrand cross-links in FA-A cells.

DNA interstrand cross-links induced by psoralen are not repaired in mammalian mitochondria.

Although it is generally known that mitochondria are defective in DNA damage processing, little is known about the DNA repair pathways and mechanisms that exist in these vital organelles. Certain lesions that are removed by base excision repair are efficiently removed in mitochondria, whereas some bulky lesions that are removed by nucleotide excision repair are not repaired in these organelles. There has been much interest in whether mitochondria possess activities for recombination repair, and some previous studies have reported such activities, whereas others have not. We have taken the approach of studying the formation and removal of interstrand cross-links (ICLs) in DNA. These lesions are thought to be repaired by a repair mechanism that involves nucleotide excision and recombinational repair. The formation and repair of DNA ICLs by 4'-hydroxymethyl-4,5',8-trimethylpsoralen was investigated in both the nuclear and mitochondrial genomes in hamster cells. Seven-fold-higher levels of ICLs were generated in mtDNA than in the dihydrofolate reductase gene, clearly indicating that the mitochondrial genome is a preferential target of 4'-hydroxymethyl-4,5',8-trimethylpsoralen damage. ICLs were removed efficiently from the dihydrofolate reductase gene, but no repair was observed in mtDNA. Our observations support previous work showing efficient gene-specific repair of these lesions in the nucleus but suggest that repair of this type of ICL does not exist in the mitochondria. The preferential damage of mtDNA and the absence of cross-link repair further suggests that mtDNA may be a biologically important target for psoralen.

Effect of mimosine on DNA synthesis in mammalian cells.

We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.

Identification of the template-binding cleft of T7 RNA polymerase as the site for promoter binding by photochemical cross-linking with psoralen.

We describe a novel method of photo-cross-linking DNA-binding proteins to DNA employing psoralen as a tether. We apply this method for the interaction of T7 RNA polymerase to its promoter. The crystallographic model of T7 RNA polymerase shows a cleft formed by the palm, thumb, and fingers domains. It was proposed that template DNA binds in the cleft. Here we directly and positively identify, in solution, the cleft as the seat of template binding. We photo-cross-linked a 23 bp promoter DNA to T7 RNA polymerase. We then determined the masses of cross-linked tryptic peptides by mass spectrometry and analyzed their amino acid composition. The cross-linked peptides were projected on the crystal structure of T7 RNA polymerase. The peptides nicely decorated the back, front, and side wall of the cleft. In a previous work [Sastry et al. (1993) Biochemistry 32, 5526-5538] we used site-specific psoralen furan-side monoadducts for cross-linking DNAs to DNA-binding proteins. We cross-linked a single-stranded 12-mer oligonucleotide to T7 RNA polymerase. We isolated and purified a DNA cross-linked tryptic peptide. We then used mass spectrometry and amino acid composition analysis to identify the location of this peptide on the T7 RNA polymerase primary sequence. In the present work we have mapped this peptide on the 3-D structure of T7 RNA polymerase. This peptide maps in the fingers domain of the polymerase. On the basis of a comparison of the map positions of peptides that cross-linked to either promoter DNA or single-stranded oligo-DNA, we propose that different functional domains may be involved in binding of double-stranded promoter DNA and nonspecific single-stranded DNA. Whereas the cleft of the polymerase is the seat of double-stranded promoter binding, the fingers domain may be used by the polymerase to grab single-stranded DNA (or RNA) in a nonspecific manner. Alternatively, the single-stranded oligo binding site may be an RNA product-binding site during transcription. The photochemical techniques we have developed [Sastry et al. (1993) Biochemistry 32, 5526-5538; this work] can be applied to other DNA-protein complexes to map DNA-binding domains.

In vivo binding of trimethylpsoralen detects DNA structural alterations associated with transcribing regions in the human beta-globin cluster.

In order to increase our knowledge about the mechanisms that regulate expression of human beta-like globin genes, we have used a novel technique to analyze the chromatin structure in living cells. This approach allowed us to detect specific DNA regions in vivo where nucleosome folding or unconstrained DNA supercoiling in erythroid cells differs from that in non-erythroid cells. In this method, we use 4,5',8-trimethylpsoralen (TMP) as a probe capable of detecting altered chromatin conformations. Our results show that TMP binds to DNA with a higher affinity over the regions in the locus that are actively expressed, including both the promoter and the transcribed region. This higher affinity detected when comparing erythroid cells with non-erythroid cells does not extend to other regions inside the beta-globin cluster. Our data suggest that the observed effect is likely due to nucleosome displacement. Alternatively, it could result from localized DNA supercoiling, but not from widespread torsional stress across the entire beta-like globin locus as hypothesized previously.

8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis.

The distribution and repair of 8-methoxypsoralen-DNA interstrand cross-links in the ribosomal RNA genes (rDNA) in Tetrahymena thermophila have been studied in vivo by Southern blot analysis. It is found that the cross-links at a density of < or = 1/2 x 10(4) base pairs (bp) are distributed equally between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis. Finally, it is shown that the cross-links in the rDNA molecules are repaired at equal rate in all three domains within 24 h and that RNA synthesis is partly restored during this repair period. The majority of the cells also go through one to two cell divisions in this period but do not survive.

Mutagenic processing of psoralen monoadducts differ in normal and Fanconi anemia cells.

The molecular spectra of mutations photoinduced (405 nm) by 4,5',8-trimethylpsoralen monoadducts (MA), at an endogenous locus, hypoxanthine-guanine phosphoribosyl-transferase (HPRT) in normal and in a Fanconi anemia (FA) lymphoblast cell line, complementation group D, are presented. We show that, in normal cells, MA induce only base substitutions. In contrast, in FA cells which are partially deficient in the incision of MA, deletions are preferentially induced over point mutations (62% of the total). Although the proportion of base substitutions is lower in FA cells, their type and sequence distribution are similar in FA and normal cell lines. The majority of base substitutions are located at sites of psoralen MA which suggest that 4,5',8-trimethylpsoralen photoinduced mutations are targeted and preferentially formed in the non-transcribed strand. Moreover, point mutations induced by MA in normal and FA cells are not homogeneously distributed, they preferentially occur in exon 8 of the HPRT gene. This heterogeneous distribution of mutations is ascribed to processing of MA. Great similarities were found between normal and FA cells with respect to the nature and location of point mutation at the HPRT gene; the high proneness to deletions remains one of the major instability features of FA.

Laser-induced protein-DNA cross-links via psoralen furanside monoadducts.

We have developed a technique for cross-linking DNA binding proteins to DNA using psoralen furanside monoadducts as photoaffinity probes and a continuous-wave argon ion laser (366 nm) as a light source. Several DNA binding proteins (T7 RNA polymerase, UvrB, single-stranded DNA binding protein of Escherichia coli, T4 gp32, and RecA of E. coli) are shown to cross-link to single-stranded psoralen monoadducted DNA oligos differing in length and sequence. Increasing fluences of laser light on a fixed ratio of DNA/protein resulted in an increase in the yield of cross-links. Titration experiments were carried out to measure the apparent cross-linking constant (KappXL) for T7 RNA polymerase or UvrB to a monoadducted 24 mer DNA. The estimated values for the apparent cross-linking constant were in the range of (2-3) x 10(-7) M for both T7 RNA polymerase and UvrB. The efficiency of cross-linking was investigated as a function of the length of adducted DNA and also as a fraction of the total noncovalent binding of proteins of psoralenated DNAs. The results showed that in the cases of T7 RNA polymerase and UvrB cross-linking was more efficient with short oligos (8 and 19 mers) as compared to longer oligos (50 mer). A tryptic peptide of T7 RNA polymerase that was conjugated to a psoralen furanside monoadducted 12 mer DNA was isolated by high-performance liquid chromatography. Mass spectrometry and amino acid composition of this peptide revealed that it originated from a region between residues 558 and 608 of the primary structure of T7 RNA polymerase. Two other peptides cross-linked to oligos were also purified. Repeated attempts to perform Edman sequencing of the peptide-DNA conjugates failed. Overall evidence indicates that photo-cross-linking of furanside monoadducts occurred at multiple sites on the proteins. We have shown that T7 RNA polymerase molecules in a ternary complex arrested at the furanside monoadduct can be cross-linked to the DNA templates with laser light. Evidence suggests that the arrested polymerase molecules existed in multiple conformations on the DNA template. This method of transcriptional cross-linking offers a new method for preparing highly stable elongation complexes for further studies.

Skin concentration of 8-methoxypsoralen, 5-methoxypsoralen and 4,5,8-trimethylpsoralen in guinea pigs.

The skin concentrations of 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and 4,5,8-trimethylpsoralen (TMP) were studied in the albino guinea pig following oral administration and intraperitoneal injection. The skin concentration of phototoxic drugs after oral administration peaked at 1.5 h, and the concentration of 8-MOP was 3.5 times greater than that of 5-MOP. The skin concentration of TMP was not detected in our study (limit of sensitivity 5 ng/ml). The highest skin concentrations of 8-MOP and 5-MOP after intraperitoneal injection were achieved for both drugs at 0.5 h, with the level of 8-MOP approximately 1.3 times higher than that of 5-MOP. The level of TMP could not be measured even in the case of intraperitoneal injection.

Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link. Differential effects of superhelical density and comparison of preincision complexes.

The effect of negative supercoiling on UvrABC incision of covalently closed duplex DNA circles containing either a furan-side monoadduct or a cross-link of 4'-hydroxymethyl-4,5',8-trimethylpsoralen at a unique site was examined. The rate of UvrABC incision of these DNA substrates was measured as a function of superhelical density, sigma, for values of sigma between 0 and -0.050. The monoadducted DNA substrate was incised at close to the maximum rate at all superhelical densities, with only a slight stimulation of activity between sigma = 0 and -0.035. In contrast, efficient UvrABC incision of the cross-linked DNA substrate required the DNA to be underwound, and activity showed a linear dependence on superhelical density up to sigma = -0.035. DNase I protection studies show that in the presence of both UvrA and UvrB a protein complex binds to the site of a psoralen monoadduct or cross-link in linear DNA. This UvrA-UvrB-dependent complex binds with similar affinity to both the monoadducted and the cross-linked DNA helices. However, differences in the DNase I footprint on these two DNA substrates indicate that the interaction of this protein complex is different at these two lesions. The addition of UvrC to linear DNA molecules that are saturated at the site of the lesion with the UvrA-UvrB-dependent complex resulted in efficient nicking of the monoadducted DNA, but not the cross-linked DNA. Thus, the properties of a DNA lesion site that lead to UvrAB recognition and binding are not necessarily sufficient to allow incision when all three Uvr subunits are present. We propose that after recognition and binding of a lesion site by the UvrAB complex and prior to incision, the damaged DNA helix undergoes a conformational change such as unwinding or melting that is induced by the lesion-bound Uvr complex.

Intercalation of psoralen into DNA of plastid chromosomes decreases late during barley chloroplast development.

We have used a DNA crosslinking assay to measure intercalation of the psoralen derivative HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into barley (Hordeum vulgare) plastid chromosomal DNA during chloroplast and etioplast development. Intercalation into DNA in intact plastids in vivo and in plastid lysates in vitro shows that chromosomal DNA in the most mature chloroplasts intercalates HMT less efficiently than DNA in younger chloroplasts. In contrast, there is no change in HMT intercalation during etioplast differentiation in the dark. Our results also show that DNA in higher plant plastid chromosomes is under superhelical tension in vivo. The lower susceptibility to HMT intercalation of DNA in the most mature chloroplasts indicates that late during chloroplast development the superhelical tension or the binding of proteins to the DNA or both change.

Light affects the structure of Chlamydomonas chloroplast chromosomes.

We have analyzed changes in the structure of chloroplast chromosomes in response to light in growing Chlamydomonas cells using a crosslinking assay based on the intercalation of HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into DNA. Our results show that the structure of chloroplast chromosomes in at least three widely separated regions is different in light-grown vs. dark-grown cells. Structural changes in chloroplast chromosomes occur within 3 hrs after exposure to light or darkness, respectively. The response to light is not inhibited by atrazine and can be elicited by dim blue light incapable of evolving O2, indicating that it does not require photosynthesis. Inhibition of cytoplasmic protein synthesis with cycloheximide prevents this response to light, indicating that it depends, at least in part, on proteins imported from the cytoplasm.

Dark and photoreactivity of 4'-aminomethyl-4,5',8-trimethylpsoralen with T7 phage.

The dark and photoreactions of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of view. The dark reaction leads to the structural destabilization of the double helix of the DNA as is shown by optical melting measurements. The genotoxicity of AMT in the dark is comparable with that of known genotoxic drugs as determined by phage inactivation. The photoreaction with UVA light leads to the formation of mono- and di-adducts depending on the wavelength and dose used. Mono- and di-adducts influence DNA stability differently; biologically both types of adducts are genotoxic as measured by action spectra.

Effects of nuclear isolation on psoralen affinity for chromatin.

We have tested the effects of nuclear isolation on intercalation of TMP (a psoralen) at specific sequences and in total DNA of cultured human cells. DNA in nuclei photobound about 20% more TMP than in cells and about 10% as much as purified DNA. In contrast, a transcribed ras gene and a randomly selected polymorphic sequence each bound about 20% more TMP than total DNA in cells. However, in nuclei, as in purified DNA, both sequences were just as sensitive as total DNA. Apparently, chromatin in cells exists within diverse TMP-binding environments and some of this diversity was lost upon nuclear isolation.