Bispecific, Cleavable, Protein DNA Crosslinker, Psoralen-Diol-Nitroveratrole. A Probe of Bacteriophage Structure

Citation

Begley, Tadhg Pulcarious (1983) Bispecific, Cleavable, Protein DNA Crosslinker, Psoralen-Diol-Nitroveratrole. A Probe of Bacteriophage Structure. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/BPQM-KP51. https://resolver.caltech.edu/CaltechTHESIS:10202010-084119498

Abstract

The synthesis of a cleavable, photochemically activated, bispecific protein-DNA crosslinking reagent, psoralen-diol-nitroveratrole (PDN) is described. The reagent crosslinks the capsid proteins to the packaged DNA in bacteriophage T7. The SDS dissociated crosslinked phage appears under the electron microscope as a rosette with the phage head at the center. DNA from the crosslinked phage does not enter agarose gels in the absence of SDS. Treatment of the crosslinked phage with proteinase K or cleavage of the crosslink with sodium periodate restores the gel mobility of the DNA to that of the non-crosslinked phage DNA. No evidence for protein-protein crosslinking was obtained when the protein composition of crosslinked and non-crosslinked phage was compared by polyacrylamide gel electrophoresis. No evidence for DNA-DNA crosslinking was obtained when the frequency of crossed BglI restriction fragments for the crosslinked and non-crosslinked phage was compared by electron microscopy. An attempt was made to analyze the distribution of protein crosslinked to intraphage DNA. It was not possible to carry out this analysis by electron microscopy as the non-crosslinked phage gave too high a background of phage heads attached to the DNA. Sodium periodate treatment of the crosslinked DNA-protein complex failed to give detectable levels of protein on a silver stained polyacrylamide gel.

The phage head of bacteriophage λ was also crosslinked to the DNA by treatment with PDN and irradiation at long wavelength (> 360 nm). The crosslink was cleaved by sodium periodate. However, proteinase K treatment did not remove the protein from the DNA. Evidence is presented indicating that the λ phage head is exceptionally stable to dissociation. Preliminary crosslinking results are presented for simian virus 40.

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