John E. Hearst

The reaction of the psoralens with deoxyribonucleic acid

Psoralen photochemistry is specific for nucleic acids and is better understood at the molecular level than are all other methods of chemical modification of nucleic acids. These compounds are used both for in vivo structure analysis and for photochemotherapy since they easily penetrate both cells and virus particles. Apparently, natural selection has selected for membrane and virus penetrability during the evolution of these natural products. Most cells are unaffected by relatively high concentrations of psoralens in the absence of ultraviolet light, and the metabolites of the psoralens have thus far not created a problem. Finally, psoralens form both monoadduct and cross-links in nucleic acid helices, the yield of each being easily controlled by the conditions used during the photochemistry.

EFFICIENT FORMATION OF A CROSSLINKABLE HMT MONOADDUCT AT THE Kpn I RECOGNITION SITE

Abstract The double‐stranded Kpn I linker, CGGTACCG, is readily crosslinked by 4′‐hydroxymethyl‐4,5′,8‐trimethylpsoralen (HMT). Under identical conditions, the corresponding Bam HI linker, CGGATCCG, is resistant to modification. The differential reactivity of HMT towards the two sequences is also observed with SV40 DNA where the Kpn I restriction site is ten‐fold more susceptible to HMT modification than the Bam HI site. Selective reaction with the Kpn I site is attributed to preferential intercalation of HMT into the TpA sequence. The availability of crosslinked Kpn I linker has allowed us to characterize the kinetics of photoreversal. Upon irradiation at 254 nm, the diadduct reverses at a faster rate than the monoadduct, leading to an accumulation of the latter. The resultant monoadduct reforms crosslink rapidly upon irradiation at 365 nm. DNA oligomers like the Kpn I linker, which can be modified by the above protocol to contain a crosslinkable HMT monoadduct, could he exploited as photoreactive sequence‐specific probes.

Structure of a psoralen-thymine monoadduct formed in photoreaction with DNA☆

Abstract A photoreaction product between calf thymus DNA and 8-methoxypsoralen, a naturally occurring psoralen, was hydrolyzed with acid to isolate a major species of the reaction, 8-methoxypsoralen-thymine monoadduct. The crystal structure of the monoadduct has been determined. The structure confirms the results of other spectroscopic studies and allows one to conclude or suggest that: (1) the photoreaction occurs preferentially at both T-A and A-T sequences in natural DNA; (2) the photocrosslink introduces a substantial kink in the DNA structure; (3) the configuration of the photoproduct is “cis-syn” at both ends of the psoralen; (4) the thymine and psoralen moieties remain planar even after the photoreaction; and (5) the angle between psoralen and thymine in the photoproducts is not fixed but has a limited range of flexibility.

RESISTANCE OF THE SCRAPIE AGENT TO INACTIVATION BY PSORALENS

–In searching for a nucleic acid within the scrapie agent, we employed psoralens which penetrate the coats of most conventional viruses, form photoadducts with their genomes, and block replication of the viruses. Five psoralens, at concentrations up to 500 times greater than those required to inactivate conventional viruses, did not influence scrapie agent titers in partially purified preparations from murine spleen and hamster brain. 3H‐psoralens were used to monitor the formation of photoadducts within nucleic acid standards added to preparations of the scrapie agent. Since no inhibition of psoralen photoadduct formation was observed in these preparations, one of three possibilities seems likely: the scrapie agent is devoid of nucleic acid, the psoralens failed to penetrate the protein coat of the agent, or its nucleic acid is unreactive with psoralens.

Identification of a structural hairpin in the filamentous chimeric phage M13Gori1

Abstract M13G ori 1 is a filamentous chimeric bacteriophage constructed in vitro from the replicative forms of two single-stranded DNA viruses by the insertion of a 2216-base fragment of G4 viral DNA into the intergenic region of M13 (Kaguni & Ray, 1979). The cloned insert contains a functional G4 origin of complementary strand synthesis (ori C ). We have tested the model in which either the M13 ori C or the G4 ori C in M13G ori 1 is located at the end of the virion. By photochemically crosslinking the DNA strands in situ with hydroxymethyl trioxsalen, we determined that the chimeric DNA is oriented with the most stable M13 hairpin, which is presumably non-essential for replication, at one end of the virus. This is the same hairpin as that found at the end of the closely related wild-type fd virion. The M13 ori C and G4 ori C are, respectively, 2333 and 1244 bases away from the end hairpin in the chimeric phage. The G4 insert is between the M13 ori C and this most stable hairpin located at the end of the virion. Thus, location of the ori C at the end of the filamentous virion is not a necessary requirement for initiation of replication. A probable role for the end hairpin in phage morphogenesis is proposed.

Interaction of psoralen derivatives with the RNA genome of Rous sarcoma virus

Abstract We have examined the interaction of several psoralen derivatives with the RNA genome of Rous sarcoma virus (RSV). In the presence of long-wavelength uv light, 4,5′,8-trimethylpsoralen (trioxsalen) and its 4′-hydroxymethyl (HMT) and 4′-aminomethyl (AMT) derivatives all efficiently crosslinked the subunits of the RSV 70 S RNA complex into a nondissociable state. These psoralen derivatives also were able to penetrate virions and crosslink the RNA complex in situ. The ability of psoralen to crosslink the subunits of viral RNA in situ is consistent with the presumption that the RSV genome exists as a complex within the virion and suggests an approach for a detailed examination of regions of secondary structure. The addition of psoralen adducts to viral RNA correlated with a loss of virus infectivity and a decrease in the amount and length of viral DNA synthesized in vitro and in vivo. Our results indicate that the RSV genome contains a significant amount of secondary structure that is reactive with psoralen compounds and that the addition of HMT to viral RNA causes premature chain termination during viral DNA synthesis.

Sedimentation of wormlike coils. II

An application of the theories of Hearst and Stockmayer for the sedimentation coefficient of wormlike coils of length shorter than 2.2 Kuhn statistical lengths, and Gray, Bloomfield and Hearst for longer wormlike coils with excluded volume to recent sedimentation data on homogeneous DNA samples is presented. The data is entirely consistent with the predictions of the theories. The molecular parameters obtained from the analysis of the data are a Kuhn statistical length at 0.2 ionic strength of 1150 A; at 0.1 ionic strength of 1290 A; and a chain backbone diameter of 20–30 A.