Evidence for structural deformation of the DNA helix by a psoralen diadduct but not by a monoadduct.

We have investigated the structural change in a double-stranded DNA helix caused by covalent addition of a psoralen. A synthetic double-stranded DNA was constructed to contain either a psoralen furan-side monoadduct or an interstrand diadduct at a specific site. When the unmodified and psoralen modified DNAs were examined by electron microscopy in the presence of distamycin, which stiffens the DNA helix, the DNA containing the psoralen interstrand diadduct appeared bent (or kinked), whereas the furan-side monoadducted DNA appeared similar to the unmodified DNA. RecA protein from E. coli has been shown to preferentially bind UV (ultra violet) irradiated DNA presumably due to alterations in the normal DNA helical structure. Using a nitrocellulose filter binding assay, we have found that the psoralen interstrand diadduct enhances the binding of recA protein to the double-stranded DNA, whereas a furan-side monoadduct has little effect. Thus both the recA protein binding and the electron microscopic data suggest that a psoralen diadduct causes deformation of a DNA helix, most likely by kinking the helix, and that a monoadduct has little effect on the DNA helix structure.     

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)     

Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.     

Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase.

The effect of apurinic/apyrimidinic (AP) sites in DNA on RNA and protein synthesis was studied in vitro using T7 coliphage DNA. Initiation of RNA synthesis by Escherichia coli RNA polymerase was synchronized and heparin was used to prevent reinitiation. When the T7 DNA contained AP sites, the rate of RNA synthesis was decreased but it remained higher than the values calculated on the assumption that an AP site in the transcribed strand is a complete block to the enzyme progression. Moreover, after the time taken by an unimpeded enzyme to go from promoter to terminator, the rate of RNA synthesis remained elevated and the number of complete RNA molecules (7000 nucleotides) continued to increase for some time. These results suggest that, if the E. coli RNA polymerase is stopped by an AP site, most often, after a pause, the enzyme resumes elongation of the RNA chain which is continuous over the AP site. Sometimes however, RNA synthesis is definitively interrupted during the pause; the probability of interruption has been estimated to be 0.3 in our experimental conditions. When a nick is placed 5' to the AP site by an AP endonuclease, the results are similar: most often, the RNA chain is synthesized without interruption past the nick in the template strand. The pause of the E. coli RNA polymerase at this combined lesion appears to be shorter than when the AP site is intact. To investigate whether a nucleotide is placed in the RNA chain in front of the AP site in the template strand by E. coli RNA polymerase, RNA synthesis was taken to completion before using this RNA for protein synthesis and measuring the activity of gene-1 product, T7 RNA polymerase. The result suggests that, after pausing, the E. coli RNA polymerase places a nucleotide in the RNA chain when passing over an AP site. The mechanism of the delayed lethality of T7 coliphages treated with monofunctional alkylating agents, which is due to the appearance of AP sites, is discussed.     

Wavelength dependence for the photoreversal of a psoralen-DNA cross-link

We report an action spectrum for the photoreversal of a psoralen cross-link joining two self-complementary DNA oligonucleotides. The cross-link was formed between two thymines (T) on opposite strands of the DNA oligomers and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT). For comparison, we also present an action spectrum for the photoreversal of the isolated diadduct T-HMT-T. The wavelength dependence for the diadduct photoreversal parallels its absorption spectrum. Both the diadduct and the cross-linked DNA can be photoreversed by exposure to light with wavelengths between 240 and 313 nm. We did not observe photoreversal at 334 nm or above. At least two distinct absorption bands appear to contribute to photoreversal. We measured a quantum yield of 0.16 for photoreversal of the isolated diadduct at wavelengths between 240 and 266 nm. For wavelengths above 280 nm, the quantum yield is 0.30. We also observed a preferential photoreversal at the furan end of the psoralen in the T-HMT-T diadduct. In contrast, the cross-linked DNA oligonucleotides preferentially photoreversed at the pyrone end of the psoralen adduct. The rate constant for photoreversal of the cross-linked DNA is larger than that for the isolated diadduct at wavelengths below 300 nm.