Interhelical DNA-DNA Crosslinking of Bacteriophage Lambda: Bis(monoazidomethidium)octaoxahexacosanediamine and Bis(psoralen)nonaethyleneoxy ether, Probes of Packaged Nucleic Acid

Citation

Mitchell, Mark Allen (1983) Interhelical DNA-DNA Crosslinking of Bacteriophage Lambda: Bis(monoazidomethidium)octaoxahexacosanediamine and Bis(psoralen)nonaethyleneoxy ether, Probes of Packaged Nucleic Acid. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/mtd8-tb22. https://resolver.caltech.edu/CaltechETD:etd-09112006-153134

Abstract

A strategy for determining the structure of packaged bacteriophage DNA is developed. The synthesis and characterization of two new interhelical crosslinking reagents, bis(monoazidomethidium)-octaoxahexacosanediamine (BAMO) (13) and bis(psoralen)nonaethyleneoxy ether (BPNE) (7) are reported. Both BAMO and BPNE are capable of crosslinking neighboring DNA helices within intact bacteriophages λ and T7. BAMO crosslinks packaged bacteriophage λ DNA at low binding levels (r = 0.01 BAMO/bp) and can be photolyzed quickly (0.5-1 hr) with light of low energy (λ > 400 nm) to yield 17% crosslinked structures as observed under the electron microscope after isolation and restriction cleavage of the crosslinked DNA. BPNE yields 10% crosslinked structures when incubated for three hours with bacteriophage λ at a binding ratio of r = 0.28 BPNE/bp, photolysed for five hours (λ = 365 nm), followed by isolation and restriction digestion of the crosslinked DNA.

Methodology necessary to uniquely locate the site of crosslinking along the bacteriophage genome has also been developed. Directional measurements of restriction fragments have been carried out on photomicrographs of fragments labeled specifically at one end with electron dense avidin spheres. The avidin spheres are attached to the restriction fragments by incorporating a biotin containing nucleotide (Bio-dUTP) into the restriction fragment using T4 polymerase under standard labeling conditions. Subsequent restriction cleavage generates fragments labeled at one specific end. Incubation of avidin spheres with these biotinylated restriction fragments results in avidin sphere binding specifically to the labeled end of the fragments.

We have begun to generate interhelical nearest neighbors maps from the measured crosslink positions for bacteriophages λ and T7. Models have been constructed for packaged T7 DNA according to the coaxial solenoid and coil of coils models discussed in the literature. Hypothetical interhelical nearest neighbors maps from these models have been prepared.

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