Divide a Plasmid DNA Molecule into Two Independent Superhelical Domains by Sequence-Specific DNA-Binding Proteins: A DNA Superhelical Barrier Model

Abstract

Both prokaryotic and eukaryotic chromosome are organized into many independent topological domains. These topological domains are presumably formed through constraining each DNA end from rotating by the interaction with nuclear proteins, i.e., DNA-binding proteins. However, so far, there is no direct evidence to support this hypothesis. In this study, we utilized two new in vitro methods, developed in our laboratory to examine whether certain sequence-specific DNA-binding proteins can separate a plasmid DNA molecule into different DNA superhelical domains. Our new methods are based on the successful construction of several plasmid DNA templates that contain many tandem copies of one DNA-binding sites in two different locations (, B., Xiao, Y., Liu, C., Li, C., and Leng, F. (2010) Nucleic Acids Resesearch, 38, 3643-3654). Using these new methods we discovered that several sequence-specific DNA-binding proteins, i.e., LacI, GalR, AraC, λ O protein, can divide a plasmid DNA molecule into two independent superhelical domains. These independent superhelical domains are thermodynamically stable. Interestingly, CRP (E. coli cAMP receptor protein), a DNA-bind and -bending protein, cannot divide the plasmid DNA molecule into different DNA topological domains. Our results can be explained by a superhelical barrier model of nucleoprotein complexes in which DNA supercoils may be confined in localized regions. We propose that the DNA superhelical barriers are certain nucleoprotein complexes that contain stable toroidal supercoils assembled from DNA looping or tightly wrapping DNA around DNA-binding proteins. The biological significance of the new superhelical barrier model will be discussed. This work is supported by a NIH grant 5SC1HD063059-02.