The cationic porphyrins TMPyP4 and TMPyP2 possess similar structures but have strikingly different potencies for telomerase inhibition. To rationalize this difference, the interactions of TMPyP4 and TMPyP2 with an antiparallel quadruplex DNA were investigated. A single-stranded DNA oligonucleotide (G4A) containing four human telomere repeats of GGGTTA has been designed to form an intramolecular quadruplex DNA and was confirmed to form such a structure under 100 mM KCl by a DNA ligase assay, DMS footprinting, and CD spectrum analysis. By carrying out UV spectroscopic studies of the thermal melting profiles of G4A−porphyrin complexes, we provide evidence that TMPyP4 and TMPyP2 both stabilize quadruplex DNA to about the same extent. A photocleavage assay was used to determine the precise location for TMPyP4 and TMPyP2 in their interactions with quadruplex DNA. The results show that TMPyP4 binds to the intramolecular quadruplex DNA by stacking externally to the guanine tetrad at the GT step, while TMPyP2 binds predominantly to the same G4 DNA structure via external binding to the TTA loop. We propose that the inability of TMPyP2 to bind to the G4A by stacking externally to the guanine tetrad accounts for the differential effects on telomerase inhibition by TMPyP4 and TMPyP2.

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The T-antigen-induced structural changes of the SV40 replication origin were probed with three DNA-reactive antitumor agents: (+)-CC-1065, bizelesin, and pluramycin. (+)-CC-1065 is an N3 adenine minor groove alkylating agent that selectively reacts with AT-rich DNA sequences with a bent conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a straight DNA conformation. Pluramycin is an intercalative guanine alkylator whose reactivity is increased by unwinding and decreased by compression of the minor and/or major grooves of DNA. We show that while binding of T-antigen reduced the ability of (+)-CC-1065 to alkylate the AT tract in the SV40 replication origin, it did not interfere with bizelesin modification of the same sequence. These unexpected results suggest that when T-antigen binds to the SV40 origin the AT tract is in a straight DNA conformation. High-resolution DNase I footprinting experiments indicate that at least three helically in-phase T-antigen binding sites exist in the GC box region located immediately downstream of the AT tract. The binding of T-antigen enhances the reactivity of (+)-CC-1065 to the two 5'-AGTTA(asterisk) (the asterisk indicates the covalent bonding site) drug modification sites in the GC box region, demonstrating that these sites are in a bent conformation. In contrast, T-antigen inhibited the reactivity of pluramycin at sequences within the GC box region that are known not to bind T-antigen. These data, in combination with the DNase I footprinting results, suggest that T-antigen binding induces a conformational change in the DNA that no longer favors pluramycin intercalation. Based on our results, we propose that T-antigen binds tightly to the upstream region of the AT tract of SV40 replication origin forming double hexamers. In the downstream region, binding of T-antigen to the helically in-phase sites in the GC box region induces DNA bending in the opposite direction of the natural AT tract bending, while simultaneously transforming the naturally bent AT tract DNA into a straight conformation.