Date of Award
Chemistry and Biochemistry
To examine the dsDNA to ssDNA equilibrium in the context of a functional piece of DNA, we use the simple case of the hairpin/cmciform structure formed in the terminator of the beta-galactosidase gene in the cloning vector pBR322. The scheme for constructing the PdC-plasmids utilizes restriction nucleases. The pBR322 is first mutated in two locations (3175 and 3285) on the same strand of DNA using a PCR-based process. These locations flank the known cruciform extrusion site. The mutated plasmid is then digested by the nicking enzyme Nb.BbvCI, which introduces two cut sites on the same strand of the DNA. Next, a 200-fold excess of a synthetic oligonucleotide of identical sequence to the 113 bp fragment but containing PdC is mixed with the sample, followed by treatment with T4 ligase. The remaining closed circular DNA is then treated with DNA gyrase to introduce supercoils into the plasmid. The supercoiled DNA containing the PdC insert is purified from a 1% agaros gel. To examine whether the PdC is paired or unpaired in the structure, we chose 5 dC residues for replacement with PdC. Two of these (3195 and 3281) are in the interior of the plasmid sequence and are not cleaved by single strand nucleases. In contrast, we use three other locations to probe hairpin conformation. Both position 3219 and 3222 are strongly cleaved by single strand nucleases while 3216 is weakly cleaved. These correspond to the loop and stem of one hairpin of the cruciform. We then used steady-state fluorescence -detected circular dichroism to study the effects of sequence location of the properties of PdC. Our data 111 indicate that there are substantial spectral differences in regions of the DNA that are single-stranded compared to double-stranded. IV
Jones, Elizabeth Kelty, "The Effects of Neighboring Bases on PdC in Single-Stranded and Double-Stranded DNA" (2010). Honors Theses. 2041.