A MODEL FOR THE T-ANTIGEN-INDUCED STRUCTURAL ALTERATION OF THE SV40 REPLICATION ORIGIN BASED UPON EXPERIMENTS WITH SPECIFIC PROBES FOR BENT, STRAIGHT, AND UNWOUND DNA

The T-antigen-induced structural changes of the SV40 replication origi n were probed with three DNA-reactive antitumor agents: (+)-CC-1065, b izelesin, and pluramycin. (+)-CC-1065 is an N3 adenine minor groove al kylating agent that selectively reacts with AT-rich DNA sequences with a bent conformation; bizelesin also reacts with the minor groove of A T-rich sequences but is selective for a straight DNA conformation. Plu ramycin is an intercalative guanine alkylator whose reactivity is incr eased by unwinding and decreased by compression of the minor and/or ma jor grooves of DNA. We show that while binding of T-antigen reduced th e ability of (+)-CC-1065 to alkylate the AT tract in the SV40 replicat ion origin, it did not interfere with bizelesin modification of the sa me sequence. These unexpected results suggest that when T-antigen bind s to the SV40 origin the AT tract is in a straight DNA conformation. H igh-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 (the asterisk indicates the covalent bonding site) drug modification s ites in the GC box region, demonstrating that these sites are in a ben t conformation. In contrast, T-antigen inhibited the reactivity of plu ramycin at sequences within the GC box region that are known not to bi nd T-antigen. These data, in combination with the DNase I footprinting results, suggest that T-antigen binding induces a conformational chan ge 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 hexam ers. In the downstream region, binding of T-antigen to the helically i n-phase sites in the GC box region induces DNA bending in the opposite direction of the natural AT tract bending, while simultaneously trans forming the naturally bent AT tract DNA into a straight conformation.