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.     

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.     

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.     

Analysis of RNA secondary structure by photochemical reversal of psoralen crosslinks.

Aminomethyltrioxsalen (AMT), a psoralen, is known to cause interstrand crosslinks in double stranded nucleic acids. We have demonstrated the photochemical reversal of this reaction, and have used this result to develop a method for identification of specific sequences which are adjacent because of RNA secondary structure formation. E. coli 5S rRNA is used as a model system. We isolated and characterized a product that is derived from the stem region of 5S RNA.     

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.     

Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms

Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with duplicated supF reporter genes, each containing an inactivating deletion, to measure HR frequencies in mammalian cells. Our results indicated that TFO-directed ICL-induced recombination frequencies were higher in the plasmids with larger distances between duplicated supF genes than with a smaller separation distance. However, the position of the ICL relative to the reporter genes did not affect HR frequencies. Recombination spectra were altered by the distance between supF copies. Although single-strand annealing (SSA) recombinants were predominant in all plasmid substrates, the plasmid with the shortest interval (60 bp) revealed a significant proportion of gene conversions (GCs). GCs occurred exclusively in the gene containing the shortest deletion, regardless of the distance between supF genes, ICL position or deletion orientation. Our analyses indicated that SSA is the predominant mechanism of ICL processing of these substrates in mammalian cells.     

Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease

Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen–modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4′-(hydroxymethyl)-4,5′,8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex ~3 bases from the 3′-side and ~9 bases from the 5′-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.     

Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells

Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.     

Homologous recombination catalyzed by human cell extracts.

Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells. We were able to obtain Neor colonies at a frequency of 2 X 10(-3). This frequency was 100 to 1,000 times higher than that obtained with no extracts. The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies. Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination. On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro.     

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.     

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.     

The formation of serine from glycine and formaldehyde by cell free extracts of Clostridium acidi-urici.

Cells of Clostridium acidi-urici which were grown in a medium containing uric acid were harvested, disrupted by sonication and centrifuged. After centrifugation the supernatant which served as the cell free extract was used to study the synthesis of serine from 2-14C glycine and formaldehyde. Serine was isolated from the reaction mixture by column chromatography. After identification by paper chromatography, serine was degraded carbon by carbon to locate the position of the labelled carbon. Radioactivity was confined almost exclusively to the alpha carbon of serine which was derived from the alpha carbon of glycine. Formaldehyde, therefore, binds at the alpha carbon of glycine to form serine. Conversion of serine to pyruvate was prevented by adding EDTA to the reaction mixture.     

The rejoining of double-strand breaks in DNA by human cell extracts.

A double-strand DNA break was introduced at a specific site within the lacZ gene of plasmid pUC18 using one of several restriction enzymes, and the plasmid exposed to nuclear extracts from human cell lines. Physical rejoining of DNA was monitored by Southern analysis after gel separation, and the fidelity of rejoining by expression of the lacZ gene after bacterial transformation with the treated plasmid. Breaks at the SalI and EcoRI sites were rejoined by extracts to form circular monomers, but the efficiency of rejoining was much higher at the SalI site. Measurement of rejoining at several adjacent sites having different types of termini, consistently showed a range of efficiencies with 5' 4-base greater than 3' 4-base overhangs and 4-base greater than 2-base greater than no overhang. Similar efficiencies were found for nuclear extracts from transformed cell lines, both from a 'normal' individual and an ataxia-telangiectasia (A-T) patient, and from a non-transformed normal cell culture. In contrast at some sites, especially those with a low rejoin efficiency, the fidelity of rejoining was very much lower for the A-T extracts than for normal cell extracts. Mis-rejoining was, however, unrelated to rejoin efficiency at other sites, suggesting that factors such as the exact sequence at the break site on the molecule may also influence the fidelity of rejoining.     

Biosynthesis of sesquiterpene alcohols and aldehydes by cell free extracts from orange flavedo

Water soluble enzymes obtained from orange flavedo form two isomeric farnesols from 1-³H labeled geranylpyrophosphate plus isopentenylpyrophosphate     

The complete pattern of mutagenesis arising from the repair of site-specific psoralen crosslinks: analysis by oligonucleotide hybridization

Psoralen crosslinks were site-specifically placed in plasmid pBR322 near the BamHI site in the tet gene by enzymatically inserting mercurated nucleotides and reacting at the target site with a sulfhydryl-containing psoralen. The damaged plasmid was repaired in SOS-induced E. coli cells. Mutants were detected by colony hybridization to oligonucleotides in the target region, and their sequences were determined. The mutations are all base substitutions, 80% transitions and 20% transversions, similar to the mutations previously identified by the loss of tetracycline resistance. However, the mutation sites detected by a physical method, unconstrained by phenotypic changes, follow a broader distribution than those identified genetically. They occur primarily at favored psoralen crosslinking sites, where T-T and T-C interstrand crosslinks can be formed. A majority of these mutations are silent.     

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.