%0 Journal Article
%T The Stability of a Model Substrate for Topoisomerase 1-Mediated DNA Religation Depends on the Presence of Mismatched Base Pairs
%A William H. Gmeiner
%A Freddie Salsbury Jr.
%A Chris M. Olsen
%A Luis A. Marky
%J Journal of Nucleic Acids
%D 2011
%I Hindawi Publishing Corporation
%R 10.4061/2011/631372
%X Topoisomerase 1 (Top1) enzymes regulate DNA superhelicity by forming covalent cleavage complexes that undergo controlled rotation. Substitution of nucleoside analogs at the +1 position of the DNA duplex relative to the Top1 cleavage site inhibits DNA religation. The reduced efficiency for Top1-mediated religation contributes to the anticancer activity of widely used anticancer drugs including fluoropyrimidines and gemcitabine. In the present study, we report that mismatched base pairs at the +1 position destabilize the duplex DNA components for a model Top1 cleavage complex formation even though one duplex component does not directly include a mismatched base pair. Molecular dynamics simulations reveal G-dU and G-FdU mismatched base pairs, but not a G-T mismatched base pair, increase flexibility at the Top1 cleavage site, and affect coupling between the regions required for the religation reaction to occur. These results demonstrate that substitution of dT analogs into the +1 position of the non-scissile strand alters the stability and flexibility of DNA contributing to the reduced efficiency for Top1-mediated DNA religation. These effects are inherent in the DNA duplex and do not require formation of the Top1:DNA complex. These results provide a biophysical rationale for the inhibition of Top1-mediated DNA religation by nucleotide analog substitution. 1. Introduction DNA topoisomerasesregulate the topological state of DNA as required to relieve superhelical density for important biological processes such as replication and transcription [1¨C3]. DNA topoisomerase 1 (Top1) is expressed at elevated levels during S-phase of the cell cycle and is the topoisomerase primarily responsible for relieving superhelical density generated in front of advancing replication forks in mammalian cells. Top1 preferentially binds superhelical DNA and forms a covalent complex as a result of nucleophilic attack by the hydroxyl of Tyr 723 on the phosphodiester backbone of the scissile strand of the DNA duplex. DNA superhelical density is reduced by controlled rotation of the scissile strand about the nonscissile strand in the cleavage complex [4, 5]. Following release of superhelical tension, the cleavage complex is dissociated by nucleophilic attack of the free 5¡ä-OH of the scissile strand to reform the phosphodiester backbone. DNA sequences that have several A-tracts flanking a conserved DNA duplex motif are also substrates for DNA Top1 and serve as a model system for understanding DNA recognition and catalysis by Top1 [6]. Top1 is the sole target for the camptothecin (CPT)
%U http://www.hindawi.com/journals/jna/2011/631372/