Psoralen Cross-linking Research Articles

In vivo mapping of nucleosomes using psoralen-DNA crosslinking and primer extension.

By the use of psoralen crosslinking and primer extension, a method was developed which allows the analysis of chromatin structure in vivo. Using a yeast minichromosome, >9 nucleosomes were mapped with a resolution of at least +/-30 bp.

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.

Chromatin structure and methylation of rat rRNA genes studied by formaldehyde fixation and psoralen cross-linking.

By using formaldehyde cross-linking of histones to DNA and gel retardation assays we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome- packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify fragments from agarose gels, corresponding to coding, enhancer and promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyse independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of Hpa II and Msp I restriction enzymes. We found CpG methylation mainly present in enhancer and promoter regions of inactive rRNA gene copies. The methylation of one single Hpa II site, located in the promoter region, showed particularly strong correlation with the transcriptional activity.

Probing the mechanisms of T7 RNA polymerase transcription initiation using photochemical conjugation of psoralen to a promoter.

We have dissected the steps in T7 RNA polymerase transcription initiation using psoralen cross-linking. DNA templates containing cross-links at either -14/-13, -2/-1, or -4/-3 were constructed. These cross-links are within the DNA-contacting region in the initiation complex. A cross-link at -2/-1 did not affect T7 RNA polymerase binding affinity, whereas a cross-link at -14/-13 reduced binding affinity by less than 2-fold. Transcription initiation was completely blocked by cross-links at -14/-13 or at -2/-1. A cross-link at -4/-3 inhibited neither binding nor the first RNA phosphodiester bond but greatly inhibited further RNA chain extension. Circular dichroism spectroscopy revealed that DNA melting in the -4/-3 cross-link was greatly inhibited, indicating that inhibition of RNA chain extension was a melting defect. Transcription shutoff on the -14/-13 cross-link may be due to inhibition of conformational changes in the polymerase-DNA complex. Because the -2/-1 cross-link is immediately upstream of the start site (+1), open complex formation may have been completely inhibited by this cross-link, accounting for the shutoff of transcription. Thus, depending on their location, psoralen cross-links affected different steps in the initiation process. We propose that promoter melting is progressive and that melting of one or two bp upstream of the +1 site is sufficient for formation of the first phosphodiester bond while further RNA chain extension within the promoter depends on greater upstream melting of the promoter, which may be required for stabilization of the initiation complex.

An unusual form of transcriptional silencing in yeast ribosomal DNA.

Generalized transcriptional repression of large chromosomal regions in Saccharomyces cerevisiae occurs at the silent mating loci and at telomeres and is mediated by the silent information regulator (SIR) genes. We have identified a novel form of transcriptional silencing in S. cerevisiae in the ribosomal DNA (rDNA) tandem array. Ty1 retrotransposons marked with a weakened URA3 gene (Ty1-mURA3) efficiently integrated into rDNA. The mURA3 marker in rDNA was transcriptionally silenced in a SIR2-dependent manner. MET15 and LEU2 were also partially silenced, indicating that rDNA silencing may be quite general. Deletion of SIR4 enhanced mURA3 and MET15 silencing, but deletion of SIR1 or SIR3 did not affect silencing, indicating that the mechanism of silencing differs from that at telomeres and silent mating loci. Deletion of SIR2 resulted in increased psoralen cross-linking of the rDNA in vivo, suggesting that a specific chromatin structure in rDNA down-regulates polymerase II promoters.

Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target.

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.

Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation.

Bacteriophage phi29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during maturation. An intriguing feature of phi29 assembly is that a virus-encoded RNA (pRNA) is required for the packaging of its genomic DNA. Psoralen cross-linking, primer extension, and T1 RNase partial digestion revealed that pRNA had at least two conformations; one was able to bind procapsids, and the other was not. In the presence of Mg2+, one stretch of pRNA, consisting of bases 31 to 35, was confirmed to be proximal to base 69, as revealed by its efficient cross-linking by psoralen. Two cross-linking sites in the helical region were identified. Mg2+ induced a conformational change of pRNA that exposes the portal protein binding site by promoting the refolding of two strands of the procapsid binding region, resulting in the formation of pRNA-procapsid complexes. The procapsid binding region in this binding-competent conformation could not be cross-linked with psoralen. When the two strands of the procapsid binding region were fastened by cross-linking, pRNA could neither bind procapsids nor package phi29 DNA. A pRNA conformational change was also discernible by comparison of migration rates in native EDTA and Mg2+ polyacrylamide gel electrophoresis and was revealed by T1 RNase probing. The Mg2+ concentration required for the detection of a change in pRNA cross-linking patterns was 1 mM, which was the same as that required for pRNA-procapsid complex formation and DNA packaging and was also close to that in normal host cells.

The SLA RNA gene of Trypanosoma brucei is organized in a tandem array which encodes several small RNAs

We have recently identified the Spliced Leader Associated RNA (SLA RNA) which is implicated in pre-messenger RNA splicing in Trypanosoma brucei by virtue of its interaction with the 5′ splice site of the trans spliced spliced leader RNA (SL RNA) in vivo. Southern analyses reveal that the SLA RNA gene is found in a tandem array of 10–11 copies per haploid genome in T. brucei. Each repeat unit in the array encodes three additional small RNAs of unknown function. RNA polymerase inhibition studies are consistent with transcription of all four genes by the same polymerase, but do not clearly differentiate between RNA polymerases II and III. The SLA RNA has homologs in other kinetoplastid protozoa and we have determined the sequence from two additional species, Trypanosoma cruzi and Crithidia fasciculata. Features of both secondary structure and sequence are conserved in these organisms. One conserved element, 5′-UGUAGUG-3′, has the potential to base-pair to the SL RNA upstream of the 5′ splice site. This potential interaction is consistent with the sites of SL RNA to SLA RNA psoralen cross-linking in vivo [1].

In vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides.

Triple helices represent an attractive method for modulating specific gene expression. In particular, cross-linking between a triplex-forming oligonucleotide (TFO) and its duplex DNA target, typically through the formation of psoralen photoadducts, allows efficient blocking of elongation by RNA polymerases in vitro. However, in vivo, this approach is limited by DNA repair of the photoadduct. Here we describe the use of an oligodeoxyribonucleotide 19mer psoralen-modified TFO to form covalent linkages between an oligonucleotide and both strands of the targeted duplex DNA, thereby efficiently blocking expression of a luciferase reporter gene. Most importantly, we demonstrate that both the psoralen cross-link and the purine-motif triplex remained intact for at least 72 h post-transfection, indicating that such species can persist for an extended period of time in vivo. These findings support the feasibility of an antigene approach for the therapeutic regulation of specific gene expression.

Highly diverged U4 and U6 small nuclear RNAs required for splicing rare AT-AC introns.

Removal of a rare class of metazoan precursor messenger RNA introns with AU-AC at their termini is catalyzed by a spliceosome that contains U11, U12, and U5 small nuclear ribonucleoproteins. Two previously unidentified, low-abundance human small nuclear RNAs (snRNAs), U4atac and U6atac, were characterized as associated with the AT-AC spliceosome and necessary for AT-AC intron splicing. The excision of AT-AC introns therefore requires four snRNAs not found in the major spliceosome. With the use of psoralen crosslinking, a U6atac interaction with U12 was identified that is similar to a U6-U2 helix believed to contribute to the spliceosomal active center. The conservation of only limited U6atac sequences in the neighborhood of this interaction and the potential of U6atac to base pair with the 5' splice site consensus for AT-AC introns provide support for current models of the core of the spliceosome.

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.

A nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5' splice site.

The mechanism by which intron-containing RNAs are recognized by the splicing machinery is only partly understood. A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has previously been shown to play a role in pre-mRNA splicing. Using a combination of splicing complex and psoralen cross-linking analysis we demonstrate that CBC is required for efficient recognition of the 5' splice site by U1 snRNP during formation of E (early) complex on a pre-mRNA containing a single intron. However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron. In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC. These results support models in which the splice sites in a pre-mRNA are originally recognized by interactions spanning exons. The defects in splicing and U1 snRNP binding caused by CBC depletion can be specifically reversed by recombinant CBC. In summary, efficient recognition of the cap-proximal 5' splice site by U1 snRNP is facilitated by CBC in what may be one of the earliest steps in pre-mRNA recognition. Data in Colot et al. (this issue) indicate that this function of CBC is conserved in humans and yeast.

Sequence-Specific Labeling of Superhelical DNA by Triple Helix Formation and Psoralen Crosslinking

Site-specific labeling of covalently closed circular DNA was achieved by using triple helix-forming oligonucleotides 10, 11 and 27 nt in length. The sequences consisted exclusively of pyrimidines (C and T) with a reactive psoralen at the 5'-end and a biotin at the 3'-end. The probes were directed to different target sites on the plasmids pUC18 (2686 bp), pUC18/4A (2799 bp) and pUC1 8/4A-H 1 (2530 bp). After triple helix formation at acid pH the oligonucleotides were photocrosslinked to the target DNAs via the psoralen moiety, endowing the covalent adduct with unconditional stability, e.g. under conditions unfavorable for preservation of the triplex, such as neutral pH. Complex formation was monitored after polyacrylamide gel electrophoresis by streptavidin-alkaline phosphatase (SAP)-induced chemiluminescence. The yield of triple helix increased with the molar ratio of oligonucleotide to target and the length of the probe sequence (27mer > 11mer). The covalent adduct DNA were visualized by scanning force microscopy (SFM) using avidin or streptavidin as protein tags for the biotin group on the oligonucleotide probes. We discuss the versatility of triple helix DNA complexes for studying the conformation of superhelical DNA.

A Novel Spliceosome Containing U11, U12, and U5 snRNPs Excises a Minor Class (AT–AC) Intron In Vitro

A minor class of introns with noncanonical splice (AT–AC) and branch site sequences exists in metazoan protein coding genes. We have established a HeLa cell in vitro system that accurately splices a pre-mRNA substrate containing such an intron from the human P120 gene. Splicing occurs via a lariat intermediate whose branch site A residue is predicted to bulge from a duplex formed with the low abundance U12 small nuclear ribonucleoprotein (snRNP), which we confirm by psoralen cross-linking. Native gel electrophoresis reveals that U11, U12, and U5 snRNPs assemble onto the P120 pre-mRNA to form splicing complexes. Inhibition of P120 splicing by 2′-O-methyl oligonucleotides complementary to U12 or U5 demonstrates that U12 and U5 snRNPs perform essential roles in the AT–AC spliceosome.

The Solution Structure of a Psoralen Cross-Linked DNA Duplex by NMR and Relaxation Matrix Refinement

The three dimensional structure of the DNA oligomer d(5′-GGGTACCC-3′)2cross-linked with 4′-aminomethyl-4,5′,8-trimethylpsoralen(AMT) has been determined by two-dimensional NMR and a relaxation matrix refinement method. NMR data and structural calculation establish that the cross-linking of the psoralen in the B-DNA duplex retains Watson-Crick type hydrogen bonding throughout the duplex, although the thymine residue which is cross-linked with psoralen forms a weaker hydrogen bond. The psoralen cross-linking in DNA duplex induces significant change of the local DNA structure, but has no important effect on overall DNA helix. Our observation places an upper limit of 10° on overall DNA bending by psoralen cross-linking, which is consistent with the result of HMT-octamer cross link [Spielmann, H. P., Dwyer, T. J., Sastry, S. S., Hearst, J. E., and Wemmer, D. E., (1995)Proc. Natl. Acad. Sci. USA92,2345–2349] and gel electrophoretic study [Haran, T. E., and Crothers, D. E. (1988)Biochemistry27,6967–6971].

Psoralen crosslinking between human immunodeficiency virus type 1 RNA and primer tRNA3(Lys).

Initiation of reverse transcription is a crucial step of retroviral infection. In HIV-1, it involves hybridization of the 18 3'-terminal nucleotides of the primer tRNA3(Lys) to the primer binding site (PBS) of the viral RNA. Moreover, additional interactions between the two RNAs were recently evidenced [Isel et al. (1995) J. Mol. Biol. 247, 25269-25272]. To get further information on the topology of the viral RNA/tRNA3(Lys) complex, we used psoralen to induce RNA-RNA crosslinking. A defined intermolecular crosslinked complex was obtained. The crosslinked regions were characterized by RNase T1 digestion followed by bi-dimensional gel electrophoresis. The crosslinked residues (nucleotide mcm5S2U34 and U35 in the anticodon loop of tRNA3(Lys) and UCU154 in the viral RNA upstream of the PBS) were mapped using a retardation method coupled with random hydrolysis. The formation of this crosslink depends on the same elements that are required for the formation of the extended interactions between primer and template RNAs, i.e., the modified bases of the tRNA and a conserved A-rich loop located upstream of the PBS in the genomic RNA. Therefore, the present crosslinking data provide relevant information on the topology of the template/primer binary complex.

Inactivation of topoisomerases affects transcription-dependent chromatin transitions in rDNA but not in a gene transcribed by RNA polymerase II.

Previous studies on a chromatin reporter gene (GAL-URARIB) in yeast showed that nucleosomes were maintained but rearranged during transcription in galactose, which was consistent with local dissociation of histones at the site of the RNA polymerase. Furthermore, repositioning of nucleosomes occurred rapidly after glucose repression. Because nucleosomal disruption and transcription produce topological changes in the chromatin substrate, the effect of topoisomerase activity was tested by the insertion of GAL-URABIB in topoisomerase mutant strains. The chromatin structure was analysed by nuclease digestion and psoralen crosslinking, and compared with that of the rDNA locus. In GAL-URARIB, neither the inactivation of topoisomerases I, II or I and II generated nucleosomal loss during transcription, nor was topoisomerase activity required for repositioning of the nucleosomes after repression. In contrast, the inactivation of topoisomerase I promoted an enhanced psoralen accessibility of the transcribed rDNA, possibly because of altered supercoiling, and the inactivation of topoisomerases I and II disrupted the chromatin structure of the whole rDNA locus by redistribution of the nucleosomes. The inactivation of topoisomerase II alone had no effect. These observations substantiate a differential participation of topoisomerases in the modulation of the chromatin structures of rDNA genes and of a single copy polymerase II gene. It is suggested that topological stress in genes transcribed by RNA polymerase II might diffuse away into flanking regions.

Site-specific substitution of inosine at the terminal positions of a pre-mRNA intron: Implications for the configuration of the terminal base interaction

Genetic evidence in yeast has revealed that a non-Watson-Crick base-pairing interaction between terminal guanosine residues of the intron is required for the second step of pre-mRNA splicing. To explore the likely configuration of the interaction between the terminal guanosines of the intron, inosine was uniformly incorporated into an adenovirus pre-mRNA substrate (Ade) to replace guanosine residues. Splicing of the inosine-containing Ade pre-mRNA was completely inhibited. Psoralen cross-linking reveals that the association of U1 and U2 snRNPs with the intron was impaired. To eliminate the deleterious effects caused by complete inosine replacement, guanosine residues at the splice site(s) of the Ade pre-mRNA were substituted by inosine. Such pre-mRNA substrates were obtained by ligation of two or three RNA fragments; the 3′ piece was primed with inosine or mono-phosphate inosine. Splicing of the Ade pre-mRNA containing an inosine residue at the 5′ or the 3′ splice site, or at both sites proceeds normally. Thus, the functions of the terminal guanosine residues of the intron in splicing can be replaced by inosine. This result supports the previous notion that an N1-carbonyl symmetric interaction likely occurs between the intron terminal residues during pre-mRNA splicing.

Genotoxic potential of psoralen cross-links versus monoadducts in normal human lymphoblasts

Using the 4,5′.8-trimethylpsoralen in combination with the reirradiation protocol, we show that, in normal human lymphoblasts, the cytotoxic potential of photoinduced cross-links (CL) is higher than that of monoadducts (MA). In contrast to cytotoxicity, the significant increase in the proportion of CL, at a constant level of total adducts, had no effect on the induction of mutations at the HPRT locus. Comparison with the data obtained in yeast and rodent cells using the same double irradiation protocol shows that the mutagenic potential of CL versus MA varies between species. This suggests that the equilibrium between the excision, the recombinational and the mutagenic components of the repair pathways which probably determine the mutagenic efficiency of CL versus MA is likely to be species-dependent.

Specialized chromatin structure domain boundary elements flanking a Drosophila heat shock gene locus are under torsional strain in vivo

An in vivo assay employing psoralen cross-linking was used to investigate the presence of unrestrained supercoiling in DNA sequences located in nontranscribed regions flanking the 3' ends of the pair of divergent heat shock protein 70 (hsp70) genes at locus 87A7 of Drosophila. Two of the regions examined contain sequences comprising the previously defined specialized chromatin structure elements (scs and scs'). Both of these putative chromosomal domain boundaries exhibited very similar levels of unrestrained negative supercoiling that remained high regardless of the transcriptional status of the hsp70 genes. The steric accessibility of the scs region before heat shock was 3-fold higher than either flanking region (consistent with its previously documented DNase I hypersensitivity); this increased an additional 2-fold following hsp70 gene activation without a concomitant rise in the accessibility of flanking regions. Most notably, a sequence which lies outside the presumed 87A7 domain, as defined by the centromere-proximal scs element, exhibited no detectable torsional tension regardless of gene activity in the domain. A sequence located just inside the scs region displayed a low level of tension that was also essentially unaffected by transcription, consistent with data obtained previously for a similarly situated fragment at the centromere-distal scs' location. The existence of a highly localized region of supercoiling within the scs and scs' sequences might be related to their activity in vivo as insulators of chromosomal position effects in Drosophila.