Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion

Psoralen intercalates into double-stranded DNA and photoreacts mainly with thymines to form monoadducts and interstrand cross-links. We used an oligonucleotide model to demonstrate a novel mechanism: the reversal of psoralen cross-links by base-catalyzed rearrangement at 90 degrees C (BCR). The BCR reaction is more efficient than the photoreversal reaction. We show that the BCR occurs predominantly on the furan side of a psoralen cross-link. The cleavage does not result in the breaking of the DNA backbone, and the thymine base freed from the cross-link by the cleavage reaction appears to be unmodified. Similarly, BCR of the furan-side monoadduct of psoralen removed the psoralen molecule and regenerated the unaltered native oligonucleotide. The pyrone-side psoralen monoadduct is relatively resistant to BCR. One can use BCR to perform efficient oligonucleotide-directed, site-specific delivery of a psoralen monoadduct. As a demonstration of this approach, we have hybridized a 19 base long oligonucleotide vehicle containing a furan-side psoralen monoadduct to a 56 base long complementary oligonucleotide target strand and formed a specific cross-link at the target site with 365-nm UV. Subsequent BCR released the oligonucleotide vehicle and deposited the psoralen at the target site.     

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.     

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.     

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)     

In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I

Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.     

The repair of psoralen monoadducts by the Escherichia coli UvrABC endonuclease.

We have examined the interactions of UvrABC endonuclease with DNA containing the monoadducts of 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP). The UvrA and UvrB proteins were found to form a stable complex on DNA that contains the psoralen monoadducts. Subsequent binding of UvrC protein to this complex activates the UvrABC endonuclease activity. As in the case of incision at pyrimidine dimers, a stable protein-DNA complex was observed after the incision events. For both 8-MOP and TMP, the UvrABC endonuclease incised the monoadduct-containing strand of DNA on the two sides of the monoadduct with 12 bases included between the two cuts. One incision was at the 8th phosphodiester bond on the 5' side of the modified base. The other incision was at the 5th phosphodiester bond 3' to the modified base. The UvrABC endonuclease incision data revealed that the reactivity of psoralens is 5'TpA greater than 5'ApT greater than 5'TpG.     

Contribution of XPF functional domains to the 5' and 3' incisions produced at the site of a psoralen interstrand cross-link.

XPF forms a heterodimeric complex with ERCC1 and is required for the repair of DNA interstrand cross-links. In association with ERCC1, it is involved in production of the 5' incision at the site of a psoralen interstrand cross-link as well as the 3' incision. The present study was carried out to determine the functional domains of XPF that are important in the production of the 5' and 3' incisions that occur at a site of a psoralen interstrand cross-link. Monoclonal antibodies (mAbs) were utilized that had been generated against polypeptide fragments of XPF and affinity-mapped to specific regions of XPF. These mAbs were examined for their ability to differentially inhibit production of dual incisions in DNA by normal human chromatin-associated protein extracts that contain XPF and ERCC1. These studies show that two regions of XPF, one N-terminal region from amino acids 12-166 and one C-terminal region from amino acids 702-854, are the most important in the production of the 5' incision. The same N-terminal region and the C-terminal region from amino acids 702-916 are also involved in the 3' incision, though to a much lesser extent. Since this C-terminal region corresponds to the proposed site of interaction of ERCC1 with XPF, these results suggest that binding of ERCC1 to XPF is critical for its ability to produce the 5' and 3' incisions at the site of an interstrand cross-link, possibly through activation or regulation of the endonucleolytic activity of the N-terminal domain of XPF.     

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.     

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.     

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.     

Photo-cross-linking of psoralen-derivatized oligonucleoside methylphosphonates to single-stranded DNA.

The preparation of oligodeoxyribonucleoside methylphosphonates derivatized with 3-[(2-aminoethyl)carbamoyl]psoralen [(ae)CP] is described. These derivatized oligomers are capable of cross-linking with single-stranded DNA via formation of a photoadduct between the furan side of the psoralen ring and a thymidine of the target DNA when the oligomer-target duplex is irradiated with 365-nm light. The photoreactions of (ae)CP-derivatized methylphosphonate oligomers with single-stranded DNA targets in which the position of the psoralen-linking site is varied are characterized and compared to results obtained with oligomers derivatized with 4'-[[N-(aminoethyl)amino]methyl]-4,5',8-trimethylpsoralen [(ae)AMT]. It appears that the psoralen ring can stack on the terminal base pair formed between the oligomer and its target DNA or can intercalate between the last two base pairs of the oligomer-target duplex. Oligomers derivatized with (ae)CP cross-link efficiently to a thymidine located in the last base pair (n position) or 3' to the last base pair (n + 1 position) of the target, whereas the (ae)AMT-derivatized oligomers cross-link most efficiently to a thymidine located in the n + 1 position. The results show that both the extent and kinetics of cross-linking are influenced by the location of the psoralen-linking site in the oligomer-target duplex.     

RecA-dependent incision of psoralen-crosslinked DNA by (A)BC excinuclease.

Previous work to elucidate the mechanism of crosslink repair by (A)BC excinuclease has shown that a psoralen-crosslinked duplex is selectively incised in the furan-side strand, while a three-stranded structure is incised in the pyrone-side strand of the crosslink. These observations support a sequential incision and recombination model for the complete error-free repair of a psoralen crosslink. The work presented here extends these findings by demonstrating that in the presence of RecA protein and a homologous DNA oligonucleotide, (A)BC excinuclease is induced to incise the pyrone-side strand of a crosslinked double-stranded plasmid molecule. This finding adds further support to the current model for error-free crosslink repair.     

Base-catalyzed reversal of a psoralen-DNA cross-link

Base-catalyzed reversal of a psoralen-DNA cross-link has been observed under denaturing alkaline conditions at elevated temperatures. The cross-link was formed between 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen and the two thymidine residues (T) on opposite strands of the double-stranded DNA formed from the self-complementary oligonucleotide 5'-GGGTACCC-3'. In contrast to the photoreversal of the cross-link, which yields mostly the furan-side monoadducted oligonucleotide [Cimino, G. D., Shi, Y., & Hearst, J. E. (1986) Biochemistry 25, 3013-3020], base-catalyzed reversal of the cross-link yields only pyrone-side monoadducted oligonucleotides as identified on the basis of their mobilities on a 20% polyacrylamide-7 M urea gel and their chemical and photochemical properties. A mechanism has been proposed to explain the base-catalyzed reversal reaction. This observation suggests a way to make pyrone-side monoadducted DNA. It also suggests that caution must be taken when psoralen-adducted DNA is treated under denaturing alkaline conditions.     

High resolution psoralen mapping reveals an altered DNA helical structure in the SV40 regulatory region.

Preferential psoralen photobinding sites have been mapped in vitro on restriction fragments spanning the SV40 origin region and surrounding sequences by a new fine structure analysis technique. Purified DNA fragments were photoreacted with 3H-5-methylisopsoralen (3H-5-MIP), a psoralen derivative which forms only monoadducts. Fragments were then end-labeled and digested with lambda exonuclease, a 5' processive enzyme which we have determined pauses at 5-MIP monoadducts. When photobinding sites were mapped on denaturing sequencing gels, it was observed that 5-MIP binds preferentially to 5'-TA sites, and to a lesser degree to 5'-AT sites. Utilizing this approach, we have identified a psoralen hypersensitive region in which the binding sites were much stronger than those in the surrounding sequences. This region extends from 150 base pairs (bp) to the late side of the enhancers to the early enhancer/promoter boundary. We suggest that this region contains a sequence directed structural alteration of the DNA helix which can be detected by the psoralen mapping approach described.     

Strand specificity of mutagenic bypass replication of DNA containing psoralen monoadducts in a human cell extract.

Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origin-dependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4'-hydroxymethyl-4,5',8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250- to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZalpha reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T.A-->C.G transitions, transversions at C.G base pairs, and deletions of single A.T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis.     

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.