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A model for the T-antigen-induced structural alteration of the SV40 replication origin based upon experiments with
1Drug Dynamics Institute, College of Pharmacy, University of Texas at Austin 78712-1074, USA.
Abstract:
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.
Insights
SV40 T-antigen binding to the replication origin alters DNA structure. It straightens the AT tract while bending the GC box, impacting drug interactions and revealing new insights into viral DNA replication mechanisms.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- The SV40 replication origin is a crucial DNA sequence for viral DNA replication.
- Simian virus 40 (SV40) T-antigen is a viral protein essential for initiating DNA replication.
- Understanding the structural dynamics of the SV40 replication origin upon T-antigen binding is key to deciphering replication mechanisms.
Purpose of the Study:
- To investigate the structural changes induced by SV40 T-antigen binding at the SV40 replication origin.
- To probe the DNA conformation using sequence-selective DNA-reactive antitumor agents.
- To elucidate the role of DNA structure in T-antigen-mediated replication initiation.
Main Methods:
- Utilized three DNA-reactive antitumor agents: (+)-CC-1065, bizelesin, and pluramycin, with distinct DNA binding specificities.
- Analyzed drug-agent reactivity at the SV40 replication origin in the presence and absence of T-antigen.
- Employed high-resolution DNase I footprinting to map T-antigen binding sites.
Main Results:
- T-antigen binding did not prevent bizelesin modification of the AT tract, suggesting a straight conformation.
- T-antigen binding enhanced (+)-CC-1065 alkylation in the GC box, indicating a bent conformation in this region.
- T-antigen binding inhibited pluramycin intercalation in the GC box, consistent with induced structural changes.
Conclusions:
- SV40 T-antigen binding induces a significant conformational change at the replication origin, straightening the AT tract and bending the GC box region.
- These structural alterations are critical for the initiation of SV40 DNA replication.
- The findings provide a detailed model of T-antigen-DNA interactions and their impact on origin DNA structure.