| (+)-CC-1065 and bizelesin are DNA-reactive anticancer agents that can also be used as structural probes of DNA. In the first project of this study, they were utilized to probe the DNA conformational changes induced by T-antigen, a multifunctional viral protein, using the in vitro SV40 DNA replication system. We show that while binding of T-antigen reduced the ability of (+)-CC-1065 to alkylate the AT tract of the SV40 replication origin, it did not interfere with bizelesin modification of the same sequence. These results suggest that T-antigen binding to the SV40 origin changes the DNA conformation of the naturally bent AT tract into a straight form. Subsequent studies show that this T-antigen-mediated straightening effect is facilitated by the flanking domains of the AT tract.; This project was further carried into an in vivo study, in which the biological consequence of bizelesin damage was evaluated. By transfecting COS-7 cells with the {dollar}beta{dollar}-gal reporter gene that was specifically modified by bizelesin at the AT tract, which also serves as the TATA box of the SV40 early promoter, we demonstrate that bizelesin damage almost completely shuts down the expression of the {dollar}beta{dollar}-gal gene.; Porphyrins and their metal complexes are photosensitive DNA-cleaving agents, which have been proposed to inhibit tumor growth by targeting telomerase. In the second project of this study, interactions of certain cationic porphyrins with plasmid and G-quadruplex DNA were investigated. A preliminary guideline for designing better porphyrin-based telomerase inhibitors that are less DNA-reactive was proposed. We show that metal ions, positive charges, and steric hindrance of the side chains all affect the photocleavage activity of a specific porphyrin. Using a newly developed photocleavage assay in combination with other biochemical and biophysical techniques, we provide direct evidence that the strikingly different potencies for telomerase inhibition by porphyrin compounds TMPyP4 and TMPyP2 can be related to their distinct binding modes to the G-quadruplex DNA. |
