Photoreactivities and thermal properties of psoralen cross-links

We have studied the photoreaction of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP), and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) with a pair of 18-base-long oligonucleotides in which a 14-base region is complementary. Only one 5'TpA site, favored for both monoadduct and cross-link formation with psoralen, is present in this oligonucleotide pair. We have used this model system to demonstrate, for the first time, strand specificity in the photoreaction of psoralen with DNA. We found that the two types of cross-links which form at this site have large differences in thermal stabilities. In addition, the denaturation of each cross-link isomer duplex occurred in at least three stages, which can be visualized as three bands in thermal equilibrium under the conditions of a denaturing polyacrylamide gel. This novel observation suggests that there are several domains differing in thermal stability in a psoralen cross-link.     

Evidence for structural deformation of the DNA helix by a psoralen diadduct but not by a monoadduct.

We have investigated the structural change in a double-stranded DNA helix caused by covalent addition of a psoralen. A synthetic double-stranded DNA was constructed to contain either a psoralen furan-side monoadduct or an interstrand diadduct at a specific site. When the unmodified and psoralen modified DNAs were examined by electron microscopy in the presence of distamycin, which stiffens the DNA helix, the DNA containing the psoralen interstrand diadduct appeared bent (or kinked), whereas the furan-side monoadducted DNA appeared similar to the unmodified DNA. RecA protein from E. coli has been shown to preferentially bind UV (ultra violet) irradiated DNA presumably due to alterations in the normal DNA helical structure. Using a nitrocellulose filter binding assay, we have found that the psoralen interstrand diadduct enhances the binding of recA protein to the double-stranded DNA, whereas a furan-side monoadduct has little effect. Thus both the recA protein binding and the electron microscopic data suggest that a psoralen diadduct causes deformation of a DNA helix, most likely by kinking the helix, and that a monoadduct has little effect on the DNA helix structure.     

Thermostability of double-stranded deoxyribonucleic acids: effects of covalent additions of a psoralen

We have carried out a thermodynamic study on the effects of covalent additions of the psoralen derivative HMT, 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, on the stability of double-stranded deoxyoligonucleotides. This was done with two systems. The first was a double-stranded DNA formed by two non-self-complementary oligonucleotides, 5'-GAAGCTACGAGC-3' and 5'-GCTCGTAGCTTC-3', where we site specifically placed an HMT molecule on the thymidine residue in oligonucleotide 5'-GAAGCTACGAGC-3' as either a furan-side monoadduct or a pyrone-side monoadduct. The second was a double-stranded DNA formed by a self-complementary oligonucleotide, 5'-GGGTACCC-3', where we placed an HMT molecule on the thymidine residue of each strand as a furan-side monoadduct or cross-linked the two strands with an HMT molecule linked to the two thymidines. We found that HMT cross-linking of the two strands stabilizes the double helix formed by 5'-GGGTACCC-3', as one might expect. Less predictable results were that the monoaddition of a psoralen stabilizes the double helix formed by the two non-self-complementary oligonucleotides by as much as 1.3 kcal/mol as a furan-side monoadduct and 0.7 kcal/mol as a pyrone-side monoadduct at 25 degrees C in 50 mM NaCl. In contrast, the monoaddition of a psoralen on each of the two thymidines in the double helix formed by 5'-GGGTACCC-3' destabilizes the helix by 1.8 kcal/mol at 25 degrees C in 1 M NaCl. This destabilization arises from an unfavorable enthalpy change (8.6 kcal/mol) and a favorable entropy change (23 cal/K X mol) due to the two HMT molecules at the centers of each strand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae

Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.     

Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.     

DNA base composition determines the specificity of UvrABC endonuclease incision of a psoralen cross-link

The sequences flanking a psoralen interstrand cross-link may determine how it is repaired. Our comparison of the Escherichia coli UvrABC endonuclease incision of a variety of specific cross-link sequences in a single natural DNA fragment showed that DNA base composition determines which of two cross-linked DNA strands will be incised. G/C enrichment of the region 6-12 bases 5' of the modified T on the furan-side strand results in preferential incision of the furan-side strand. When the G/C-rich region is on the 3' side, or on neither side, incisions occur on either strand. These effects of DNA base composition suggest that UvrAB can bind in two ways to a psoralen cross-link.     

Construction of DNA substrates modified with psoralen at a unique site and study of the action mechanism of ABC excinuclease on these uniformly modified substrates

Psoralens bind to DNA noncovalently and upon exposure to near UV (320-400 nm) light produce covalent adducts. Thymidine residues in DNA, especially those at 5'-TpA-3' sequences, are most susceptible to the photochemical reaction. This property of the reaction and the recent advances in oligonucleotide synthesis and separation has enabled us to construct DNA fragments containing psoralen adducts at a specific site. The octanucleotide 5'-TCGTAGCT-3' was photoreacted (in the presence of the complementary strand) with the synthetic psoralen 4'-hydroxymethyl-4,5',8-trimethylpsoralen to obtain oligonucleotides adducted via the furan or pyrone ring at the internal thymine. These modified octanucleotides were ligated to nonmodified oligonucleotides to obtain a 40-base pair DNA fragment containing a psoralen adduct at a central location. The modified fragment having the thymine-furan side 4'-hydroxymethyl-4,5',8-trimethylpsoralen adduct was irradiated with 360 nm of light to produce an interstrand cross-link, and this cross-linked DNA was purified to homogeneity. These uniquely modified DNAs were used as substrates for Escherichia coli ABC excinuclease to determine its incision mechanism unambiguously and to determine the contact sites of the enzyme. ABC excinuclease mediates the cleavage of the 8th and 5th phosphodiester bonds 5' and 3', respectively, to psoralen monoadducts, and the 9th (5') and 3rd (3') phosphodiester bonds to the furan-side thymine of the cross-link. Preliminary DNaseI footprinting studies show that ABC excinuclease protects the whole 40-base pair fragment from DNaseI, and binding of the A and B subunits to the furan side-monoadducted substrate produces two hypersensitive phosphodiester bonds in the vicinity of the 5' incision site of ABC excinuclease.     

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.     

Processing of targeted psoralen cross-links in Xenopus oocytes.

Psoralen cross-links have been shown to be both mutagenic and recombinagenic in bacterial, yeast, and mammalian cells. Double-strand breaks (DSBs) have been implicated as intermediates in the removal of psoralen cross-links. Recent work has suggested that site-specific mutagenesis and recombination might be achieved through the use of targeted psoralen adducts. The fate of plasmids containing psoralen adducts was evaluated in Xenopus oocytes, an experimental system that has well-characterized recombination capabilities and advantages in the analysis of intermediates in DNA metabolism. Psoralen adducts were delivered to a specific site by a triplex-forming oligonucleotide. These lesions are clearly recognized and processed in oocytes, since mutagenesis was observed at the target site. The spectrum of induced mutations was compared with that found in similar studies in mammalian cells. Plasmids carrying multiple random adducts were preferentially degraded, perhaps due to the introduction of DSBs. However, when DNAs carrying site-specific adducts were examined, no plasmid loss was observed and removal of cross-links was found to be very slow. Sensitive assays for DSB-dependent homologous recombination were performed with substrates with one or two cross-link sites. No adduct-stimulated recombination was observed with a single lesion, and only very low levels were observed with paired lesions, even when a large proportion of the cross-links was removed by the oocytes. We conclude that DSBs or other recombinagenic structures are not efficiently formed at psoralen adducts in Xenopus oocytes. While psoralen is not a promising reagent for stimulating site-specific recombination, it is effective in inducing targeted mutations.     

Wavelength dependence for the photoreactions of DNA-psoralen monoadducts. 2. Photo-cross-linking of monoadducts

The photoreactions of HMT [4'-(hydroxymethyl)-4,5',8-trimethylpsoralen] monoadducts in double-stranded DNA have been studied with complementary oligonucleotides. The HMT was first attached to the thymidine residue in the oligonucleotide 5'-GAAGCTACGAGC-3' as either a furan-side monoadduct or a pyrone-side monoadduct. The HMT-monoadducted oligonucleotide was then hybridized to the complementary oligonucleotide 5'-GCTCGTAGCTTC-3' and irradiated with monochromatic light. In the case of the pyrone-side monoadducted oligonucleotide, photoreversal was the predominant reaction, and very little cross-link was formed at all wavelengths. The course of the photoreaction of the double-stranded furan-side monoadducted oligonucleotide was dependent on the irradiation wavelength. At wavelengths below 313 nm, both photoreversal and photo-cross-linking occurred. At wavelengths above 313 nm, photoreversal of the monoadduct could not be detected, and photo-cross-linking occurred efficiently with a quantum yield of 2.4 X 10(-2).     

Wavelength dependence for the photoreactions of DNA-psoralen monoadducts. 1. Photoreversal of monoadducts

We have studied the wavelength dependence for the photoreversal of a monoadducted psoralen derivative, HMT [4'-(hydroxymethyl)-4,5',8-trimethylpsoralen], in a single-stranded deoxyoligonucleotide (5'-GAAGCTACGAGC-3'). The psoralen was covalently attached to the thymidine residue in the oligonucleotide as either a furan-side monoadduct, which is formed through the cycloaddition between the 4',5' double bond of the psoralen and the 5,6 double bond of the thymidine, or a pyrone-side monoadduct, which is formed through the cycloaddition between the 3,4 double bond of the psoralen and the 5,6 double bond of the thymidine. As a comparison, we have also investigated the wavelength-dependent photoreversal of the isolated thymidine-HMT monoadducts. All photoreversal action spectra correlate with the extinction spectra of the isolated monoadducts. In the case of the pyrone-side monoadduct, two absorption bands contribute to the photoreversal with a quantum yield of 2 X 10(-2) at wavelengths below 250 nm and 7 X 10(-3) at wavelengths from 287 to 314 nm. The incorporation of the monoadduct into the DNA oligomer had little effect upon the photoreversal rate. For the furan-side monoadduct at least three absorption bands contribute to the photoreversal. The quantum yield varied from 5 X 10(-2) at wavelengths below 250 nm to 7 X 10(-4) at wavelengths between 295 and 365 nm. In contrast to the case of the pyrone-side monoadduct, the incorporation of the furan-side monoadduct into the DNA oligomer reduced the photoreversal rate constant at wavelengths above 285 nm.     

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.     

Induction of DNA replication-mediated double strand breaks by psoralen DNA interstrand cross-links

The effect of DNA interstrand cross-links (cross-links) on DNA replication was examined with a cell-free SV40 origin-dependent DNA replication system. A defined template DNA with a single psoralen cross-link and the SV40 origin of replication was replicated by HeLa cell-free extract in the presence of SV40 large T antigen. The psoralen cross-link inhibited DNA replication by terminating chain elongation at 1-50 nucleotides before the cross-linked sites. The termination of DNA replication by the cross-links mediated the generation of double strand breaks near the cross-linked sites. These results are the first biochemical evidence of the generation of double strand breaks by DNA replication.     

Photochemical production of psoralen - DNA monoadducts capable of subsequent photocrosslinking.

Irradiation of 4' -aminomethyl-4,5' ,8-trimethyl psoralen in the presence of DNA at wavelengths between 380 and 400nm leads to efficient production of monoadducts with no significant amount of crosslinking. The yield of monoadduct attainable is high (about 35 adducts per 1000 base pairs) and can be made still higher by repeated irradiation with fresh psoralen derivative. Subsequent irradiation at 350nm results in crosslinking of a almost half of the monoattached psoralen. Studies of the yield of nonoadduct and crosslink as function of irradiation wavelength show that the action spectrum for crosslink formation is blue-shifted relative to that for monoadduct formation.     

Repair of triple helix directed psoralen adducts in human cells.

Triple helix forming oligonucleotides can direct DNA damaging agents at specific sites in an intact double helix. In our study, triple helix formation was demonstrated in a SV40 based shuttle vector treated with psoralen linked to a 22-mer purine rich oligonucleotide. UVA irradiation caused a covalent linkage of the oligonucleotide through the psoralen to the mutational supF marker gene of the plasmid. After passage in the Jurkat human cell line the recovered vector was analysed in an indicator bacterial strain and mutants were collected. The presence of adducts in the target sequence did not reduce the yield of replicated progeny vector molecules, indicating repair of triple helix associated monoadducts and cross-links. Mutations were highly targeted to a six nucleotide long region of the target sequence. The number of target sequence mutants obtained after triple helix directed psoralen treatment was approximately 160 times higher than with free psoralen. A further investigation of the exact mechanism of the mutational process could make triple helix directed mutagenesis a more useful tool in gene therapy, antiviral therapy, and in studies on DNA repair and genome organisation.     

Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease

Interstrand DNA cross-link damage is a severe challenge to genomic integrity. Nucleotide excision repair plays some role in the repair of DNA cross-links caused by psoralens and other agents. However, in mammalian cells there is evidence that the ERCC1-XPF nuclease has a specialized additional function during interstrand DNA cross-link repair, beyond its role in nucleotide excision repair. We placed a psoralen monoadduct or interstrand cross-link in a duplex, 4-6 bases from a junction with unpaired DNA. ERCC1-XPF endonucleolytically cleaved within the duplex on either side of the adduct, on the strand having an unpaired 3' tail. Cross-links that were cleaved only on the 5' side were purified and reincubated with ERCC1-XPF. A second cleavage was then observed on the 3' side. Relevant partially unwound structures near a cross-link may be expected to arise frequently, for example at stalled DNA replication forks. The results show that the single enzyme ERCC1-XPF can release one arm of a cross-link and suggest a novel mechanism for interstrand cross-link repair.     

Specificity of site directed psoralen addition to RNA.

We describe the attachment of a psoralen derivative (site specific psoralen, SSP) to the 5' end of a DNA oligonucleotide and the hybridization and the photoreaction of this reagent with a complementary target site on an RNA molecule. SSP was coupled to a variety of DNA oligonucleotides to investigate the structural requirements for addition to the RNA. Efficient SSP photoadducts were made on specific uridines by designing an intercalation site at an unpaired nucleotide in the RNA strand within the heteroduplex region. The optimal location for this site was five nucleotides from the oligonucleotide 5' end and just 5' to the target uridine residue. Because the attachment of the SSP to the oligonucleotide is through a disulfide bond, the DNA oligonucleotide can be removed with reduction to leave SSP attached to the RNA strand. The SSP adduct made in this way will be useful for subsequent biochemical and biophysical experiments.     

Interstrand psoralen cross-links do not introduce appreciable bends in DNA

Analysis of the X-ray crystallographic structure of an 8-methoxypsoralen-thymine monoadduct has led to the suggestion that psoralen cross-links would bend DNA by as much as 70 degrees [Peckler, S., Graves, B., Kanne, D., Rapoport, H., Hearst, J. E., & Kim, S.-H. (1982) J. Mol. Biol. 162, 157-172]. DNA can exist in a stably bent configuration in solution as recently demonstrated from analysis of polyacrylamide gel electrophoresis and differential decay of birefringence. Using these techniques, we have investigated the structure of DNA cross-linked with 8-methoxypsoralen and 4,5',8-trimethylpsoralen. The results are not consistent with cross-links introducing any appreciable stable bend in double-stranded DNA molecules. Results suggest that photobound 4,5',8-trimethylpsoralen molecules lengthen DNA by the equivalent of about one base pair per photobound adduct. We have also determined that 4,5',8-trimethylpsoralen cross-links are introduced preferentially into 5'-TA compared to 5'-AT DNA sequences.