Formation by bifunctional furocoumarins of DNA crosslinks and their repair in cultured mouse embryo fibroblasts

Formation of crosslinks in DNA by three bifunctional psoralen derivatives plus UVA light in mouse embryo fibroblasts was evaluated by a NaI density gradient centrifugation method. Psoralen was shown to be a more active cross-linking agent than 8-methoxypsoralen. As for 4,5',8-trimethylpsoralen, it needed much lower concentrations and much less 365 nm light fluence to yield high percentages of crosslinked DNA. Repair of adducts formed by these psoralen derivatives was studied by splitting the irradiation dose into two equal parts separated by variously long dark repair periods. It was shown that essentially only monoadducts formed during the first irradiation period were repaired. These mouse embryo fibroblasts seem unable to repair interstrand DNA crosslinks.     

Crosslinking studies on the organization of the 16 S ribosomal RNA within the 30 S Escherichia coli ribosomal subunit.

The location and frequency of RNA crosslinks induced by photoreaction of hydroxymethyltrimethylpsoralen with 30 S Escherichia coli ribosomal subunits have been determined by electron microscopy. At least seven distinct crosslinks between regions distant in the 16 S rRNA primary structure are seen in the inactive conformation of the 30 S particle. All correspond to crosslinked features seen when the free 16 S rRNA is treated with hydroxymethyltrimethylpsoralen. The most frequently observed crosslink occurs between residues near one end of the molecule and residues about 600 nucleotides away to generate a loop of 570 bases. The size and orientation of this feature indicate it corresponds to the crosslinked feature located at the 3′ end of free 16 S rRNA.When active 30 S particles are crosslinked in 5 mm-Mg²⁺, six of the seven features seen in the inactive 30 S particle can still be detected. However, the frequency of several of the features, and particularly the 570-base loop feature, is dramatically decreased. This suggests that the long-range contacts that lead to these crosslinks are either absent or inaccessible in the active conformation. Crosslinking results in some loss of functional activities of the 30 S particle. This is consistent with the notion that the presence of the crosslink that generates the 570-base loop traps the subunit in an inactive form, which cannot associate with 50 S particles.The arrangement of the interacting regions crosslinked by hydroxymethyltrimethylpsoralen suggests that the RNA may be organized into three general domains. A striking feature of the Crosslinking pattern is that three of the seven products involve regions near the 3′ end of the 16 S rRNA. These serve to tie together large sections of rRNA. Thus structural changes at the 3′ end could, in principle, be felt through the entire 30 S particle.     

DNA polymerase eta reduces the gamma-H2AX response to psoralen interstrand crosslinks in human cells

DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase eta are sensitive to psoralen photoadducts created under conditions favoring crosslink formation, suggesting a role for translesion synthesis in crosslink repair. Because crosslinks can lead to double-strand breaks, we monitored phosphorylated H2AX (gamma-H2AX), which is typically generated near double-strand breaks but also in response to single-stranded DNA, following psoralen photoadduct formation in XP-V fibroblasts to assess whether polymerase eta is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced gamma-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase eta in XP-V cells normalized the gamma-H2AX response. In response to psoralen crosslinking, gamma-H2AX as well as 53BP1 formed coincident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced gamma-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase eta is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase eta are involved in processing crosslinks and avoiding gamma-H2AX associated with double-strand breaks and single-stranded DNA in human cells.     

Structure of E. coli 16S RNA elucidated by psoralen crosslinking

E. coli 16S RNA in solution was photoreacted with hydroxymethyltrimethylpsoralen and long wave ultraviolet light. Positions of crosslinks were determined to high resolution by partially digesting the RNA with T1 RNase, separating the crosslinked fragments by two-dimensional gel electrophoresis, reversing the crosslink, and sequencing the separated fragments. This method yielded the locations of crosslinks to +/- 15 nucleotides. Even finer placement has been made on the basis of our knowledge of psoralen reactivity. Thirteen unique crosslinks were mapped. Seven crosslinks confirmed regions of secondary structure which had been predicted in published phylogenetic models, three crosslinks discriminated between phylogenetic models, and three proved the existence of new structures. The new structures were all long range interactions which appear to be in dynamic equilibrium with local secondary structure. Because this technique yields direct information about the secondary structure of large RNAs, it should prove invaluable in studying the structure of other RNAs of all sizes.     

In vitro crosslinking of DNA by 8-methoxypsoralen visualized by electron microscopy

By electron microscopic visualisation of totally denatured DNA, we have detected photochemically induced 8-methoxypsoralen crosslinks in vitro after irradiation at 360 nm. The amount of crosslinks was expressed as the percentage of DNA length which was kept in double-stranded appearance by closely situated crosslinks. This percentage correlated well with irradiation time, irradiation intensity, and the concentration of 8-methoxypsoralen. These parameters have also been correlated with the mean size and the size distribution of non-crosslinked regions of DNA, so called bubbles. For a comparison with another psoralen type, we have carried out a similar set of experiments using 4,5,8-trimethylpsoralen.     

Human MLH1 protein participates in genomic damage checkpoint signaling in response to DNA interstrand crosslinks, while MSH2 functions in DNA repair

DNA interstrand crosslinks (ICLs) are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA) is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSα and MutSβ mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents.     

Base-pairing between distant regions of the Escherichia coli 16 S ribosomal RNA in solution.

Intramolecular crosslinks have been introduced into Escherichia coli 16 S ribosomal RNA in aqueous solution by irradiation in the presence of hydroxymethyl-trimethylpsoralen. When the crosslinked RNA is denatured and examined in the electron microscope the most striking features are a variety of large open loops. In addition, because the crosslinked molecules are shortened compared to non-crosslinked molecules, there are likely to be small hairpins not resolved by the present technique. The sizes and positions of 11 loop classes have been determined and oriented on the molecule. The frequency of occurrence of the different classes of loops depends on the crosslinking conditions. When the crosslinking is done in solutions containing Mg²⁺, at least four of the loop classes appear with greater frequency than they do in 3.5 mm-NaCl. The loops presumably arise because complementary sequences separated by long intervening regions are being crosslinked. These base-pairing interactions between residues distant in the primary structure appear to be prominent features of the secondary structure of rRNA in solution.     

Sensitivity of mitomycin C and nitrogen mustard crosslinks to extreme alkaline conditions

DNA-DNA crosslinks in cells treated with mitomycin C, nitrogen mustard, or decarbamoyl mitomycin C were measured in alkaline isopycnic gradients as a function of pH. Crosslinks from cells treated with mitomycin C and nitrogen mustard, which react with DNA purines, could be detected at pH 12.5 but not at pH 14. No crosslinks from cells treated with decarbamoyl mitomycin C were detected at either pH. Previous studies with cells exposed to psoralen derivatives plus 360 nm light, which produce DNA-DNA crosslinks with pyrimidines, demonstrated stable crosslinks at pH 14. These studies indicate that DNA-DNA crosslinks involving DNA purines are much less stable at high pH than those involving pyrimidines, and that methods involving exposure to extreme alkaline conditions may give inaccurate information for some agents.     

Gel electrophoretic technique for separating crosslinked RNAs. Application to improved electron microscopic analysis of psoralen crosslinked 16 S ribosomal RNA

We have developed a gel electrophoresis technique for separating crosslinked RNA molecules into a series of discrete fractions. The gel used is polyacrylamide made in formamide and low salt designed to denature the RNA during electrophoresis. The mobility depends upon the position of crosslinking within each molecule, as demonstrated by electron microscopy of RNA eluted from the gel. In general, molecules with large loops electrophorese more slowly than molecules with small loops or uncrosslinked molecules. We have used this technique to re-examine the psoralen crosslinking pattern of Escherichia coli 16 S ribosomal RNA in inactivated 30 S ribosomal subunits. To determine the correct orientation of each type of crosslink, we have covalently attached DNA restriction fragments to the RNA so that the polarity of the RNA in the microscope would be known. Our previous major conclusions are confirmed: the predominant long-distance crosslink detected by gel electrophoresis involves a residue close to the 3′ end and a residue approximately 600 nucleotides away: the formamide/polyacrylamide gel is able to separate two closely spaced 1100-nucleotide interactions beginning close to the 3′ end, which were reported as one interaction before: and an interaction joining the ends is detected as before. However, one low-frequency crosslinked interaction, between positions 950 and 1400, and possibly another low-frequency interaction, between positions 550 and 870, are determined to be in the opposite polarity to that described previously.     

Testing models of the arrangement of DNA inside bacteriophage lambda by crosslinking the packaged DNA

Bis-psoralens can make crosslinks between two adjacent segments of a condensed DNA molecule. We have used bis-psoralen crosslinking as a covalent means of preserving structural features of DNA packaged inside bacteriophage λ. A single bis-crosslink prevents normal electron microscopic spreading of intact λ DNA: after deproteinization the molecules appear as tangled rosettes which are presumably due either to trapped knots or supercoils. However, restriction nuclease digestion of the crosslinked DNA yields fragments that spread normally. The location of crosslinks can be studied by their appearance in such a digest as X-shaped molecular features. Significant crosslinking frequencies are found between all six possible pairs of the four largest BglII fragments of λ DNA. Little or no evidence is seen for crosslinked loops within individual fragments. These results are inconsistent with two previously suggested models of intraphage DNA packaging. Determination of the positions of crosslinks within restriction fragments yields a pattern of DNA contacts too complex for any simple analysis. The finding of hints of periodicity in the sites of crosslinks, preferential crosslinking of some restriction fragments, and the occurrence of one particularly efficient crosslinking reaction between two restriction fragments appear to rule out purely random packaging arrangements.     

Removal of psoralen monoadducts and crosslinks by human cell free extracts.

Human cell free extracts are capable of carrying out damage-induced DNA synthesis in response to DNA damage by UV, psoralen, and cisplatin. We show that this damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is 'repair synthesis' and not an aberrant DNA synthesis reaction potentiated by DNA deformed by adducts. By comparing the denaturable fraction of psoralen adducted DNA which becomes labeled in the repair reaction to that of terminally labeled DNA (without repair) we have found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach we have obtained preliminary evidence that this in vitro system is capable of removing psoralen crosslinks as well.     

gamma-H2AX formation in response to interstrand crosslinks requires XPF in human cells

To further define the molecular mechanisms involved in processing interstrand crosslinks, we monitored the formation of phosphorylated histone H2AX (gamma-H2AX), which is generated in chromatin near double strand break sites, following DNA damage in normal and repair-deficient human cells. Following treatment with a psoralen derivative and ultraviolet A radiation doses that produce significant numbers of crosslinks, gamma-H2AX levels in nucleotide excision repair-deficient XP-A fibroblasts (XP12RO-SV) increased to levels that were twice those observed in normal control GM637 fibroblasts. A partial XPA revertant cell line (XP129) that is proficient in crosslink removal, exhibited reduced gamma-H2AX levels that were intermediate between those of GM637 and XP-A cells. XP-F fibroblasts (XP2YO-SV and XP3YO) that are also repair-deficient exhibited gamma-H2AX levels below even control fibroblasts following treatment with psoralen and ultraviolet A radiation. Similarly, another crosslinking agent, mitomycin C, did not induce gamma-H2AX in XP-F cells, although it did induce equivalent levels of gamma-H2AX in XPA and control GM637 cells. Ectopic expression of XPF in XP-F fibroblasts restored gamma-H2AX induction following treatment with crosslinking agents. Angelicin, a furocoumarin which forms only monoadducts and not crosslinks following ultraviolet A radiation, as well as ultraviolet C radiation, resulted only in weak induction of gamma-H2AX in all cells, suggesting that the double strand breaks observed with psoralen and ultraviolet A treatment result preferentially following crosslink formation. These results indicate that XPF is required to form gamma-H2AX and likely double strand breaks in response to interstrand crosslinks in human cells. Furthermore, XPA may be important to allow psoralen interstrand crosslinks to be processed without forming a double strand break intermediate.     

Covalent crosslinks introduced via a triple helix-forming oligonucleotide coupled to psoralen are inefficiently repaired.

Triple helix-forming oligonucleotides (TFOs) represent potentially powerful tools to artificially modulate gene activity. In particular, they can be used to specifically introduce a lesion into a selected target sequence: interstrand crosslinks and monoadducts can be introduced via TFOs coupled to psoralen. The efficiency of these strategies depends on the cell ability to repair these lesions, an issue which is still controversial. Here we show, using psoralen-coupled TFOs and the yeast as a convenient cellular test system, that interstrand crosslinks are quantitatively poorly repaired, resulting in an efficient modification of target gene activity. In addition, these lesions result in the introduction of mutations in a high proportion of cells. We show that these mutations are generated by the Error-Prone Repair pathway, alone or in combination with Nucleotide Excision Repair. Taken together, these results suggest that TFOs coupled to psoralen could be used to inactivate a gene with significant efficiency.     

Electron microscopic identification of supercoiled regions in complex DNA structures

When intracellular lambda replicative intermediates (theta structures) are intercalated with psoralen and then irradiated with long wavelength ultraviolet light (u.v.), interstrand crosslinks are produced. After purification and denaturation of these theta structures, a global difference in denaturation can be observed by electron microscopy; parental sections are essentially native whereas daughter segments are highly denatured. This difference can be explained if parental sections are covalently continuous (and therefore able to supercoil) and daughter segments are not. Due to the higher thermal stability of supercoiled DNA, parental DNA will remain native while daughter sections will denature. Because these structures are crosslinked, the thermal treatment does not lead to dissociation of the highly denatured daughter strands. Experiments with simple negatively supercoiled plasmid circles support the above conclusions. When circles are crosslinked with psoralen-u.v. and then denatured, they remain native because of the higher thermal stability of covalently closed structures. If the circles are linearized before heating but after the psoralen-u.v. treatment, the thermal stability effect is eliminated and the molecules become highly denatured. In this case, however, the crosslinking density is found to be higher than in samples linearized before psoralen-u.v. treatment. This, therefore, shows that crosslinking density also reflects the superhelical state of the molecule at the time of psoralen-u.v. treatment. Two different properties can be used to discriminate between supercoiled and covalently discontinuous domains in complex DNA structures. First, supercoiled regions remain native while covalently discontinuous segments denature following a thermal treatment. This effect requires that covalent continuity exists up to and during the heating treatment. Second, because negative superhelicity enhances psoralen intercalation, crosslinking density is higher in these regions. Even if supercoiled domains are destroyed after the psoralen-u.v. treatment, the imprint of superhelicity is retained and can be recognized as a higher than normal crosslinking density.     

Formation of intraprotamine disulfides in vitro.

When mammalian protamine is dissolved in aqueous buffers at neutral or alkaline pH, both ends of the protein fold inward toward the center of the molecule and form disulfide crosslinks that stabilize several different structures. In the absence of reducing agents, these folded forms of protamine may be visualized and quantitated by gel electrophoresis. Using this technique, we have examined the formation of bull protamine disulfides in solution and describe a variety of factors that affect this process. At pH 8, disulfide-stabilized folded forms of protamine appear within minutes after solubilization of the fully reduced protein. Five different monomers are detected by electrophoresis. Each of these monomers is stabilized by at least one disulfide crosslink and migrates with a distinct mobility, ahead of the fully reduced and extended protein. Under certain conditions, dimers of these folded structures crosslinked by interprotamine disulfides are also formed. The appearance of these disulfide-crosslinked forms of protamine is effected by air oxidation, accelerated at alkaline pH, inhibited upon lowering the pH below pH 7 and eliminated by modifying the protein's cysteine residues. Similar intramolecular disulfides are also produced after the protamine molecule binds to DNA. These results suggest that only those cysteines located within the amino- and carboxyterminal ends of the protein appear to participate in forming intramolecular disulfides in vitro.