Binding and photoreactivity of psoralen linked to triple helix-forming oligonucleotides

Triple helix-forming oligonucleotides conjugated to a psoralen (psoTFO) have been designed to bind to three distinct purine-rich sequences within the human interstitial collagenase (MMP1) gene. Gel mobility shift assays indicate that these psoTFO bind to and photoreact with model target DNA sequences following ultraviolet A (UVA) irradiation. The dissociation constants for binding of the psoTFO to their targets range from 0.3 to 4 microM. Psoralen monoadducts with the purine-rich target strand and interstrand crosslinks are efficiently formed on targets containing either 5'-ApT-3' or 5'-TpA-3' sequences adjacent to the TFO binding sequence. The dependence of adduct formation on UVA dose has provided quantitative estimates of the overall rate constants for psoralen monoadduct and crosslink formation in the presence of a TFO. When psoralen is tethered to a TFO, the rate of monoadduct formation exceeds that of crosslinking for all sequences studied. This contrasts with the relatively low rate of monoadduct formation that has been reported for free psoralens, suggesting that the bound TFO facilitates the initial photochemistry that generates monoadducts, but does not significantly affect interstrand crosslink formation. psoTFO and UVA treatment inhibit DNA cleavage by a restriction endonuclease when the psoralen covalently reacts directly at the endonuclease site. The particular TFO studied do not completely inhibit endonuclease activity when they are noncovalently bound or when the covalent psoralen adduct does not coincide with the endonuclease site. Our findings confirm that TFO are capable of directing psoralen photoadducts to specific DNA targets and suggest that TFO can significantly modulate psoralen photoreactivity and DNA-protein interactions.     

Photobiological Origins of the Field of Genomic Maintenance

Although sunlight is essential for life on earth, the ultraviolet (UV) wavelengths in its spectrum constitute a major threat to life. Various cellular responses have evolved to deal with the damage inflicted in DNA by UV, and the study of these responses in model systems has spawned the burgeoning field of DNA repair. Although we now know of many types of deleterious alterations in DNA, the approaches for studying them and the early mechanistic insights have come in large part from pioneering research on the processing of UV‐induced bipyrimidine photoproducts in bacteria. It is also notable that UV was one of the first DNA damaging agents for which exposure was directly linked to cancer; the sun‐sensitive syndrome, xeroderma pigmentosum, was the first example of a cancer‐prone hereditary disease involving a defect in DNA repair. We provide a short history of advances in the broad field of genomic maintenance as they have emerged from research in photochemistry and photobiology.     

Reactions of 1,3,4-triphenyl-1,2-dihydrophosphete

In contrast to previously reported reactivity of the tungsten pentacarbonyl complex of a 2-substituted 1,2-dihydrophosphete, which apparently undergoes electrocyclic ring opening to the corresponding 1-phospha-1,3-butadiene and subsequent [4+2] cycloaddition reactions with dienophiles, the reaction chemistry of 1,3,4-triphenyl-1,2-dihydrophosphete is dominated by its nucleophilic nature. Although low to modest yields of cycloadducts are obtained with some dienophiles, the reactions forming these products are apparently stepwise, as indicated by the loss of stereochemistry in the reaction of dimethyl maleate and in the competitive formation of a phosphorus-free dimer in the reaction of N-methylmaleimide. Dimethyl acetylenedicarboxylate affords three major products, each of which incorporates two equivalents of the acetylene, again apparently a result of initial nucleophilic addition of the dihydrophosphete to the “dienophile.” © 1998 John Wiley & Sons, Inc. Heteroatom Chem 9:9–19, 1998     

Effects of metal coordination on the reactivity of 1,3,4-triphenyl-1,2-dihydrophosphete

Nucleophilicity dominates the reaction chemistry of 1,3,4-triphenyl-1,2-dihydrophosphete even when it is coordinated to electrophilic metal centers, but coordination dramatically alters the course of its reactions. Deoxygenation of carbonyl-containing substrates is effected by both the W(CO)₅ complex, which reductively couples benzaldehyde, and the HgCl₂ complex, which converts benzaldehyde to α,α-dichlorotoluene. Metal coordination appears to decrease the tendency of the dihydrophosphete to undergo electrocyclic ring opening to the corresponding 1-phosphabutadiene, and the HgCl₂ complex reacts with dimethyl acetylenedicarboxylate to afford a cyclopentadienyl ylide containing an intact dihydrophosphete unit. By reducing the nucleophilicity of the dihydrophosphete and/or the availability of the highly nucleophilic uncoordinated dihydrophosphete, coordination to HgCl₂ and W(CO)₅ makes accessible new mechanistic pathways. Dihydrooxaphosphinines, although unavailable through the reactions of the dihydrophosphete, may be synthesized by exploitation of the reactivity of organotitanium metallacycles. © 1998 John Wiley & Sons, Inc. Heteroatom Chem 9:21–28, 1998     

Ein erprobtes empirisches System der Klassifizierung der R?ntgen-strahlen-Beugungsaufnahmen zur identifizierung krystalliner Stoffe

Ohne Zusammenfassung     

PHOTOACI'IVATED GILVOCARCIN V INDUCES DNA-PROTEIN CROSSLINKING IN GENES FOR HUMAN RIBOSOMAL RNA AND DIHYDROFOLATE REDUCTASE

Abstract The nature of DNA interactions with photoactivated gilvocarcin V has been analyzed at the gene level in both rRNA and dihydrofolate reductase genes of human fibroblasts, utilizing a modified Southern hybridization technique. Neither interstrand DNA crosslinking nor RNA linkage to DNA was detected. However, we consistently observed in both genes retarded DNA bands appearing in a dose-dependent fashion following exposure to photoactivated gilvocarcin V. These retarded bands were enhanced when genomic DNA was prepared without proteinase K treatment, suggesting involvement of protein in this DNA interaction. Because these bands disappear following proteinase K treatment, it is probable that photoactivated gilvocarcin V induces DNA-protein crosslinking.     

ELECTROPHORETIC SEPARATION OF FUROCOUMARIN: DNA PHOTOADDUCTS

Abstract We describe a new approach for quantitating furocoumarin adducts in DNA using enzymatic hydrolysis followed by resolution and recovery of the adduct molecules by electrophoresis on polyacrylamide tube gels. The resolution of this method approaches that of high pressure liquid chromatography but at a considerably lower cost. Digestion conditions using DNase II and spleen phosphodiesterase II to yield mononucleotides and adducted bases were worked out for DNA containing 8-methoxypsoralen or 4'-hydroxymethyl-4,5',8-trimethylpsoralen. The phosphatase activity of DNase II was shown to be much more active on dNMPs than on adducted bases. This can be exploited to reduce the background of radioactivity from labeled dNMP's trailing into the adduct peaks, thereby increasing the effective sensitivity of the technique when quantitating adducts made with non-radioactive drug in labeled DNA. Since resolution of adducts requires dense (30%) acrylamide gels, we have devised a method for making extremely uniform high density gels by polymerization under static pressure. A continuous collection apparatus was constructed to recover material from gels. The identity of the resolved species was determined by several different methods, including analysis by HPLC.     

THE U.V. SENSITIVITY OF BACTERIA: ITS RELATION TO THE DNA REPLICATION CYCLE

Abstract— A striking increase in the shoulder of the u.v. survival curve but no change in the limiting slope is obtained when cultures of Escherichia coli strain TAU complete the DNA replication cycle in the absence of concommitant protein synthesis prior to irradiation. The u.v. sensitivity of protein synthesis or RNA synthesis is not altered significantly by this treatment.
In contrast to the result for strain TAU, there is no significant change in the u.v. survival curve for the u.v. sensitive E. coli B_s-1 when its DNA replication cycle is completed under similar conditions.
Following a period of inhibited protein synthesis there is a delay in the reinitiation of the normal DNA replication cycle when protein synthesis resumes. This delay would allow time for an intracellular repair system to operate before the attempted resumption of normal replication. Strain B_s-1, which is deficient in this repair system, would not be expected to benefit from such a delay, as consistent with the observed results. A model is presented to account for lethality due to attempted DNA replication during a period of repair synthesis. The maximum survival for a given u.v. dose would be predicted for a culture which has completed the normal DNA replication cycle prior to irradiation and which is not permitted to reinitiate the cycle until all possible repair synthesis is completed.     

Absence of repair replication following ultraviolet irradiation of an excision-deficient mutant of Escherichia coli

The excision -repair of damaged DNA in bacteria and other systems probably requires at least three enzymes to carry out the following steps in sequence: (1) Recognition of a structural distortion in the DNA and the production of an endonucleolytic cleavage of the damaged strand near the lesion. (2) The simultaneous peeling back of the damaged strand and resynthesis of the excised region, with eventual cleavage of the damaged segment from the DNA. (3) The rejoining of the newly synthesized strand to contiguous parental DNA. Evidence for all three steps has been obtained from in vivo studies. The E. coli DNA polymerase has been shown to carry out step # 2 in vitro [1] and the polynucleotide ligase has the required specificity for step # 3[2–4]. An enzyme responsible for step # 1 has been purified from Micrococcus lysodeikticus [5,6] but not from E. coli, although a class of u.v. sensitive mutants in E. coli has been shown to be defective in this step in the repair sequence. In such mutants the release of pyrimidine dimers from the damaged DNA is not observed during post-irradiation growth of u.v. irradiated cultures [7]. It would be predicted, as a consequence, that the next step, non-conservative repair replication, would not be seen in these mutants. Hanawalt and Petti-john showed this to be true for the double mutant E. coli B_8-1 that includes a deficiency in dimer excision [8]. In the present study we have looked more closely at an E. coli K-12 strain that has only the uvrA6 deficiency that results in inability to excise pyrimidine dimers.