Reduced 4,5',8-trimethylpsoralen cross-linking of left-handed Z-DNA stabilized by DNA supercoiling

Z-DNA-forming sequences, (GT)21, (GT)12ATGT, and (CG)6TA(CG)6, were cloned into plasmids. These sequences formed left-handed Z-DNA conformations under torsional tension from negative supercoiling of DNA. 4,5',8-Trimethylpsoralen, on absorption of 360-nm light, forms monoadducts and interstrand cross-links in DNA that exists in the B-helical conformation. Trimethylpsoralen cross-links were introduced into the potential Z-DNA-forming sequences in relaxed DNA when these sequences existed as B-form DNA. In supercoiled DNA when these sequences existed in the Z conformation, the rate of cross-linking was greatly reduced, and trimethylpsoralen did not form monoadducts appreciably to Z-DNA. As an internal control in these experiments, the rates of cross-linking of the Z-DNA-forming sequences were measured relative to that of an adjacent, cloned sequence that could not adopt a Z conformation. The initial relative rates of cross-linking to Z-DNA-forming sequences were dependent on the superhelical density of the DNA, and the rates were ultimately reduced by factors of 10-15 for Z-DNA in highly supercoiled plasmids. This differential rate of cross-linking provides a novel assay for Z-DNA. Initial application of this assay in vivo suggests that a substantial fraction of (CG)6TA(CG)6, which existed as Z-DNA in plasmid molecules purified from cells, existed in the B conformation in vivo.     

Structure of the DNA interstrand cross-link of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) cross-links a 5' TpA or a 5' ApT site by photoreacting with one thymine moiety in each DNA strand. We are interested in whether psoralen interstrand cross-links all share one structure or whether there are significant differences. In this paper, we employed a rapid method for probing the structure of the cross-link by making a series of TMP cross-linked duplexes containing specific base-pair mismatches. The relative stability provided by a base pair can be correlated with neighboring base pairs by comparing the extents of gel retardation when base-pair mismatches happen in each position. From our studies, we infer that with respect to the furan-side strand, the 5'T.A base pair of the two T.A base pairs in the TpA site is not hydrogen bonded. Immediately on each side of the cross-linked TpA site is a highly stabilized base pair. Next, a region of decreased stability occurs in each arm of a cross-linked duplex and these base pairs of least stability are located farther away from the cross-linked thymines as the lengths of the arms of the cross-linked helix increase. Finally, even in 7 M urea at 49 degrees C the cross-linked helix is hydrogen bonded at both ends of a duplex of 22 base pairs. We propose that the structures of interstrand cross-links in DNA vary appreciably with the DNA sequence, the length of the DNA duplex, and the structures of the DNA cross-linking agents.     

Inactivation of transforming Bacillus subtilis deoxyribonucleic acid by monoadducts of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) monoadducts inactive transforming deoxyribonucleic acid (DNA) in Bacillus subtilis. Contrary to TMP diadducts (TMP cross-links), which severely inhibit entry of donor DNA (G. Venema and U. Canosi, Mol. Gen. Genet. 179:1--11), TMP monoadducts have only a slight effect on entry. Since reextracted TMP-monoadduct-containing transforming DNA is a differentially repaired by Uvr- and Uvr+ recipients and cross-linkable to the recipient strand in the heteroduplex recombinant DNA molecules, the monoadducts can be integrated along with the donor DNA into the recipient chromosome.     

DNA sequence specificity of mitomycin cross-linking

Using a gel electrophoresis assay, we show that the target DNA sequence cross-linked by N-methylmitomycin A, its aziridinomitosene, and mitomycin C is CpG, in strong preference over GpC. The yield per CpG site increases as the number of successive CpG sequences increases. Molecular modeling reveals no systematic difference between the energies of mitomycin cross-links at CpG in comparison with GpC. However, the distance between guanine amino groups in CpG sequences is nearly the same as the distance in the cross-linked adduct, whereas the amino group separation at GpC sites is substantially larger in the starting DNA than in the adduct. We suggest that the favorable placement of the second reaction center in CpG greatly accelerates the second step in the cross-linking reaction. As shown by a competition assay, mitomycins bind A-T and G-C sequences noncovalently equally well, even though the only sequence that yields appreciable cross-linking is CpG. N-Methylmitomycin A and its aziridinomitosene are found to be better cross-linking agents than is mitomycin C.     

A laser flash photolysis study of the reactivities of the triplet states of 8-methoxypsoralen and 4,5',8-trimethylpsoralen with nucleic acid bases in solution.

The extinction coefficients, quantum yields and reactivities of the triplet states of 8-methoxypsoralen and 4,5',8-trimethylpsoralen in methanolic solution have been determined using laser flash photolysis techniques. The second-order rate constants for the quenching of these triplet states by pyrimidine and purine bases were found to be several orders of magnitude lower than those found for other furocoumarin derivatives. This may suggest, therefore, that the skin photosensitising ability of such compounds does not necessarily correlate with in vitro triplet state reactivity. Preliminary experiments on the reactivity of the psoralen triplet state with DNA itself indicate that no transient absorptions due to psoralen excited states can be observed when a photon is absorbed by the psoralen-DNA complex.     

Effect of 4,5'',8-trimethylpsoralen interstrand cross-links present in recipient Bacillus subtilis on the integration of transforming DNA.

When recipient Bacillus subtilis carrying chromosomal trimethylpsoralen cross-links were transformed, the donor marker activity decreased with the extent of cross-linking. Additional donor marker activity was lost upon incubation of the reextracted DNA with nuclease S1, particularly at higher levels of cross-linking. Physical analysis of the reextracted DNA showed that the donor DNA was progressively excluded from heteroduplex formation as the frequency of cross-links in the recipient DNA increased. In the donor-recipient complexes still being formed, increasing amounts of donor DNA became susceptible to nuclease S1 digestion under these conditions. These results suggest that resident interstrand cross-links interfere both with initiation of recombination and with the completion of heteroduplex formation.     

Identification of sites of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen cross-linking in Escherichia coli 23S ribosomal ribonucleic acid

The reagent 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) was used to cross-link 23S rRNA from Escherichia coli under 50S ribosomal subunit reconstitution conditions. Following partial digestion of the RNA with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate fragments derived from the cross-linked sites. These fragments were analyzed by digestion with ribonucleases T1 and A and their positions in the 23S RNA sequence identified. Fragment a1 (positions 1325-1426) is cross-linked to a2 (positions 1574-1623); fragment b1 (positions 1700-1731) is cross-linked to b2 (positions 1732-1753); and a cross-link is formed within fragment c (or c') (positions 863-916). In the latter case, the cross-link was located precisely, linking residues C867 and U913. All three HMT-mediated cross-links are consistent with a proposed secondary structure model for 23S RNA [Noller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, R. R., Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A., Gupta, R., & Woese, C. R. (1981) Nucleic Acids Res. 9, 6167-6189].     

Photosensitized inhibition of nitrate reductase induction by 4,5',8-trimethylpsoralen and near ultraviolet light

4,5' ,8-trimethylpsoralen photosensitizes the inhibition of the induction of nitrate reductase in XD cells of tobacco, by near ultraviolet light. The photosensitization depends on the concentration of 4,5' ,8-trimethylpsoralen and the near ultraviolet light dose. Concomitant with the inhibition of nitrate reductase induction there is a severe inhibition of DNA and RNA synthesis. Oxygen uptake and protein synthesis are not affected. Translation of preexisting mRNA coding for nitrate reductase is insesitive to 4,5' ,8-trimethylpsoralen and near ultraviolet light. It is concluded that the DNA of these cells is the target for the photochemical reaction of 4,5' ,8-trimethylpsoralen. The template activity of the DNA is inhibited, and the expression of the genome thereby prevented. Translation of preexisting nRNA is not affected.     

Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5',8-trimethylpsoralen

We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 microM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment. A large proportion of the label was released in alkaline sucrose gradients as a low molecular weight fraction (average length about 500 nucleotides) which was supposed to consist of replication origins containing DNA fragments. From the relative quantities of this DNA obtained at different times of the S phase we concluded that it contains a considerable but not precisely determinable proportion of non-origin DNA. We also find that the fraction is contaminated by a large excess of non-replicating bulk DNA.     

Measurement of interstrand cross-link frequency and distance between interruptions in DNA exposed to 4,5',8-trimethylpsoralen and near-ultraviolet light

Bifunctional psoralens react photochemically with DNA to form single-strand adducts and interstrand, chemical cross-links. Cross-link formation is first order with [P], the concentration of added psoralen, when [P] much less than Kd, the psoralen-DNA dissociation constant. DNA molecules containing interstrand cross-links are reversibly bihelical and so are readily detected. It was not heretofore possible to determine cross-link frequency in polydisperse DNA from the mass F of DNA spared cross-linkage. We have derived a statistical relation to calculate cross-link frequency at fixed light exposure and variable [P]. We show here that S, the initial slope of the curve described by -ln F as a function of [P], is proportional to Mw, the weight-average molecular weight of nick-free DNA. The cross-link frequency at any [P] can be determined from k, a constant measured for DNA of known Mw at low cross-linkage. This relation is valid for DNA of any molecular weight distribution. In experiments with uniform length DNA, -ln F (cross-link frequency) increased in simple proportion to [P]. Intact and restriction endonuclease HindIII digested phage lambda DNA molecules have discrete lengths. S for each was proportional to Mw of the twin helix even though the molecular weight distribution of the restriction fragments was skewed. S was proportional to Mw and to the median molecular weight of sheared cellular DNA over a wide range. Also, we found that 1/S was linear with exposure of cellular DNA to gamma radiation. S can therefore be used to calculate L, the average distance between interruptions in the double helix.     

Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light.

16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.     

Dependence of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen photoaddition on the conformation of ribonucleic acid

The photoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) to different conformational states of RNA was studied. Poly(U), poly(A,U) (random copolymer), poly(A-U) (alternating copolymer), poly(A) . poly(U) (double stranded), and poly(U) . poly(A) . poly(U) (triple stranded) were reacted with HMT at different temperatures and salt concentrations. The conformation of the polymers was monitored by UV absorption and circular dichroism. It was found that the rate of HMT photoaddition changed dramatically at structural transitions in the RNA. The alternating copolymer poly(A-U) was found to have the highest rate of addition. Low salt and temperature produced maximal incorporation.     

Frameshift mutations in bacteria produced in the dark by several furocoumarins; absence of activity of 4,5',8-trimethylpsoralen.

4 furocoumarins, namely psoralen (P), 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP) and angelicin (A) were tested for dark mutagenesis in E. coli lac-. Three compounds; P, 8-MOP and A were shown to be weak frame-shift mutagens. TMP, surprisingly in view of its very active photosensitizing action, was found to be non-mutagenic. These results are discussed in relation to the photosensitizing action of the furocoumarins.     

Comparative bacterial mutagenicity studies with 8-methoxypsoralen and 4,5',8-trimethylpsoralen in the presence of near-ultraviolet light and in the dark

2 strains of S. typhimurium, TA98 and TA100, and 2 strains of E. coli, WP2(pKM101) and WP2uvrA-(pKM101) were used to study mutagenesis by 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (4,5',8-TMP) in the dark and in the presence of near-ultraviolet (NUV) light both without metabolic activation and with rat-liver S9 at 3 levels (4, 10 and 30% in standard cofactors). The S9-independent base substitution mutagenic activity of 8-MOP plus NUV light was confirmed in WP2(pKM101), and a similar activity was seen for 4,5',8-TMP, although neither substance was active in TA100. The frameshift mutagenic activity of 8-MOP in the dark in TA98 was not confirmed despite histidine levels which would ensure DNA replication, but this may be due to the lower concentrations of 8-MOP achieved in the common solvent system adopted. Both 8-MOP and 4,5',8-TMP were mutagenic in WP2uvrA-(pKM101) after microsomal activation, and the responses were similar whether experiments were conducted in the dark or in NUV light. In view of the oral administration of 8-MOP to psoriasis patients, this finding may be of relevance in risk assessment, and tends to suggest that topical application of 4,5',8-TMP to psoriatic patients may present reduced risk of malignant disease.     

Hyperreactivity of B-Z junctions to 4,5',8-trimethylpsoralen photobinding assayed by an exonuclease III/photoreversal mapping procedure

We have developed an exonuclease III/photoreversal procedure to map, with base-pair resolution, the bases that have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand crosslinks in DNA. This assay allows quantification of relative rates of Me3-psoralen photobinding to bases in DNA at levels less than one crosslink per 8000 base-pairs. We demonstrate the applicability of the Me3-psoralen mapping procedure on the Z-forming sequence GAATT(CG)6-TA(CG)6AATTC. The results confirm our previous findings that Me3-psoralen forms crosslinks in the 5'TA within the (CG)6TA(CG)6 sequence when it exists in the B conformation but not when it exists in the Z conformation. In addition, with increasing superhelical density we observe at least a hundred-fold increased Me3-psoralen presumably represent B-Z junctions. The two presumed junctions respond differently with increasing negative superhelical tension, however, suggesting that the structures of these negative superhelical tension, however, suggesting that the structures of these junctions differ. This increased Me3-psoralen photoreactivity provides a positive signal for the presence of Z-DNA. The sequence and assay described here provide a "torsionally tuned probe" for determining the effective superhelical density of DNA in vivo.     

Mutagenesis induced by site specifically placed 4''-hydroxymethyl-4,5'',8-trimethylpsoralen adducts.

Closed circular double stranded M13mp19 DNA containing a site-specifically placed HMT (4'-hydroxymethyl-4-5'-8-trimethylpsoralen) monoadduct or crosslink was synthesized in vitro. The damaged DNA were scored for loss of infectivity by transfection into repair proficient or deficient E. coli and into SOS induced E. coli. Mutant phages were detected by the loss of alpha-complementation between the viral and the host Lac Z genes or by the acquisition of resistance to kpn I digestion. Our results indicate that HMT mutagenesis is targeted and that deletion or transversion of the modified thymidine is the predominant sequence change elicited by a monoadduct or a crosslink. Transfection of the monoadducted DNA into a Uvr A deficient strain did not change the mutation pattern but did increase the respective mutation frequencies. Transfection of the crosslinked DNA into a SOS induced host resulted in the appearence of other types of mutations attributable to an increase in both targeted and untargeted mutations.