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 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.
Han
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Drug Dynamics Institute, College of Pharmacy, University of Texas at Austin 78712-1074, USA.
Hurley
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D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology*; D000970:Antineoplastic Agents; D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000737:chemistry*; D003850:Deoxyribonuclease I; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins; D008957:Models, Genetic; D008961:Models, Structural; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D018741:Replication Origin; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics*; D014508:Urea / Q000031:analogs & derivatives
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000952:Antigens, Polyomavirus Transforming; D000970:Antineoplastic Agents; D004279:DNA, Viral; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins; D009838:Oligodeoxyribonucleotides; C003169:pluramycin; C018782:CC 1065; D014508:Urea; D003850:Deoxyribonuclease I; C072257:bizelesin