Effects of hydroxyl radical scavengers on relaxation of supercoiled DNA by aminomethyl-trimethyl-psoralen and monochromatic UVA photons.

The ability of scavengers of hydroxyl radical (OH radical) to modulate the photosensitized relaxation (induction of the first single-strand break) of supercoiled plasmid DNA with UVA photoactivated 4'-aminomethyl-4,5',8-trimethylpsoralen was examined by comparing the dose reduction factor (DRF: the ratio of fluence required to induce the same degree of relaxation in the absence to the presence of OH radical scavengers). The addition of mannitol, azide, acetate, or formate at concentrations inversely proportional to the value of the rate constants for the scavenging of OH radicals partially attenuated the supercoiled DNA relaxation. The degrees of protection afforded by the four scavengers in the presence of AMT photoactivated by either 334 nm or 365 nm monochromatic photons were similar, giving an average DRF of about 0.25 in all cases. Given the diverse chemical nature of the scavengers and their wide range of concentrations utilized, these findings are evidence for the involvement of a Type I photosensitization in the induction of DNA single-strand breaks by photoactivated AMT.

4'-Aminomethyl-4,5',8-trimethylpsoralen photochemistry: the effect of concentration and UVA fluence on photoadduct formation in poly(dA-dT) and calf thymus DNA.

The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4- and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10(5) M-1) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320-400 nm radiation) fluence.

Relaxation of supercoiled DNA by aminomethyl trimethylpsoralen and UV photons: action spectrum.

An action spectrum for the relaxation of supercoiled plasmid DNA (induction of the first single-strand break) by photoactivated 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) has been determined using monochromatic UV photons from 254 to 405 nm. The spectrum of AMT-induced plasmid DNA relaxation fits closely with the absorbance spectrum of AMT in the spectral region between 313 nm and 405 nm but deviates at wavelengths shorter than 313 nm. This assay also reveals that the psoralen photosensitization reaction with DNA also produces piperidine-labile sites. Addition of mannitol and azide partially quenches the supercoil relaxation reaction, evidence for a role of Type II photosensitization pathway.

A mutant of herpes simplex virus type 1 exhibits increased stability of immediate-early (alpha) mRNAs.

vhs1 is a herpes simplex virus type 1 mutant defective in the shutoff of both host and alpha polypeptide synthesis. In cycloheximide reversal experiments, alpha mRNAs were significantly more stable in vhs1-infected cells than in cells infected with wild-type virus, whether assayed by in vitro translation or Northern blotting.

Control of mRNA stability by the virion host shutoff function of herpes simplex virus.

vhs1 is a mutant of herpes simplex virus type 1 that is defective in the virion host shutoff function responsible for the degradation of cellular mRNAs and the concomitant shutoff of host protein synthesis. In this study, the effect of the vhs1 mutation on the metabolism of viral mRNAs was examined by measuring the half-lives and patterns of accumulation of 10 different viral mRNAs representing all kinetic classes. The vhs1 mutation had the effect of dramatically lengthening the cytoplasmic half-lives of all 10 mRNAs. In wild-type virus infections, the 10 mRNAs had similar half-lives, suggesting that little, if any, target mRNA selectivity was exhibited by the vhs function. The vhs1 mutation caused overaccumulation of a number of mRNAs. The effect was most dramatic for the alpha (immediate-early) mRNA for ICP27 and the beta (early) mRNAs encoding thymidine kinase, ICP8, and DNA polymerase. Whereas in wild-type infections these mRNAs increased to peak levels and subsequently declined in abundance, in vhs1 infections they continued to accumulate until late times. A significant but less dramatic overaccumulation was observed for several beta-gamma (delayed-early) and gamma (late) mRNAs. The results suggest that the vhs protein plays an important role in determining the half-lives of viral mRNAs belonging to all kinetic classes and in so doing is important in the normal downregulation at late times of alpha and beta gene expression.

Detection of immobilized amplicons by ELISA-like techniques.

The NucleoLink surface is a physically modified, thermostable, optically clear resin. It allows the covalent binding of 5'-phosphorylated oligonucleotides. Target DNA amplification by polymerase chain reaction (PCR) is accomplished by asymmetric amplification on the covalently immobilized primer that develops into immobilized amplicons. A DNA fragment of bovine leukemia virus is used as a model system for the detection of immobilized amplicons by ELISA-like techniques. Covalently bound oligonucleotides are also utilized as capture probe in the hybridization-based signal amplification for detection of an infectious organism.