title: Dividing a supercoiled DNA molecule into two independent topological domains.
abstract: Both prokaryotic and eukaryotic chromosomes are organized into many independent topological domains. These topological domains may be formed through constraining each DNA end from rotating by interacting with nuclear proteins; i.e., DNA-binding proteins. However, so far, evidence to support this hypothesis is still elusive. Here we developed two biochemical methods; i.e., DNA-nicking and DNA-gyrase methods to examine whether certain sequence-specific DNA-binding proteins are capable of separating a supercoiled DNA molecule into distinct topological domains. Our approach is based on the successful construction of a series of plasmid DNA templates that contain many tandem copies of one or two DNA-binding sites in two different locations. With these approaches and atomic force microscopy, we discovered that several sequence-specific DNA-binding proteins; i.e., lac repressor, gal repressor, and λ O protein, are able to divide a supercoiled DNA molecule into two independent topological domains. These topological domains are stable under our experimental conditions. Our results can be explained by a topological barrier model in which nucleoprotein complexes confine DNA supercoils to localized regions. We propose that DNA topological barriers are certain nucleoprotein complexes that contain stable toroidal supercoils assembled from DNA-looping or tightly wrapping DNA around DNA-binding proteins. The DNA topological barrier model may be a general mechanism for certain DNA-binding proteins, such as histone or histone-like proteins, to modulate topology of chromosome DNA in vivo.
authors: Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA. lengf@fiu.edu
authors: Fenfei
authors: F
authors: Leng
authors: Bo
authors: B
authors: Chen
authors: David D
authors: DD
authors: Dunlap
mesh_terms: D001665:Binding Sites; D004278:DNA, Superhelical / Q000737:chemistry*; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D056968:Lac Repressors / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; C050993:Galactose repressor proteins; D056968:Lac Repressors; D012097:Repressor Proteins