cc-1065 and pluramycin

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.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antigens, Polyomavirus Transforming; Antineoplastic Agents; Base Sequence; Binding Sites; Deoxyribonuclease I; DNA Replication; DNA, Viral; Duocarmycins; Indoles; Leucomycins; Models, Genetic; Models, Structural; Molecular Sequence Data; Oligodeoxyribonucleotides; Plasmids; Replication Origin; Restriction Mapping; Simian virus 40; Urea

Integration Host Factor (IHF) is a sequence-specific DNA-bending protein that is proposed to interact with DNA primarily through the minor groove. We have used various chemical probes [(+)-CC-1065, a minor-groove-specific agent that alkylates N3 of adenine and traps bends into the minor groove; pluramycin, a minor-major-groove threading intercalator that alkylates N7 of guanine; KMnO4, which reacts more strongly with bases in denatured DNA] to gain more information on the interaction of IHF with the H' site of phage lambda. In addition to the 13-bp core consensus recognition element present at all IHF binding sites, the H' site also has an upstream AT-rich element that increases the affinity of IHF for this site. Our results reveal new details of IHF-DNA interaction at this site. Results with (+)-CC-1065 modification suggest that IHF interacts with the adenines on the 3'-side of the AT-rich element and likely induces a minor-groove bend in its vicinity, which in turn stabilizes the interaction. Pluramycin modification experiments suggest the presence of both short- and long-range structural perturbations (possible DNA unwinding events) on either side of the IHF contact region. Although IHF is known to induce a large bend in DNA at the H' site, no separation of base pairs was detected when the bent DNA was probed with KMnO4. DNA cyclization studies indicate a large magnitude (approximately 180 degrees) for the IHF-induced bend at the H' site, consistent with > 140 degrees bend estimated by gel electrophoresis methods. These studies suggest that IHF-induced DNA bending is accompanied by the introduction of a DNA node, DNA unwinding, and/or by some other DNA distortion. An enhanced binding and stability of IHF was observed on small circular DNA.

Topics: Adenine; Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Bacteriophage lambda; Base Sequence; Consensus Sequence; DNA; DNA Footprinting; DNA-Binding Proteins; DNA, Circular; Duocarmycins; Hydroxyl Radical; Indoles; Integration Host Factors; Leucomycins; Molecular Probes; Molecular Sequence Data; Nucleic Acid Conformation; Potassium Permanganate