TY - JOUR
T1 - 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
AU - Han, Frank Xiaoguang
AU - Hurley, Laurence H.
PY - 1996/6/18
Y1 - 1996/6/18
N2 - 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 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* (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.
AB - 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 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* (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.
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U2 - 10.1021/bi960251d
DO - 10.1021/bi960251d
M3 - Article
C2 - 8672503
AN - SCOPUS:0345298454
SN - 0006-2960
VL - 35
SP - 7993
EP - 8001
JO - Biochemistry
JF - Biochemistry
IS - 24
ER -