In this paper researchers have devised a very clever strategy to evaluate the genesis of topological domains. The basic idea is to design high affinity DNA binding sites for specific proteins as tandem copies within a single plasmid. Then, they tested whether it was possible to divide the plasmid into protein defined domains of constrained topological segments (or create topological barriers). This is a nice inroad to understand how topological domains can be propagated in linear chromosomes in eukaryotes. In addition, the work describes for the first time a tractable mechanism to create and test protein/DNA defined topological domains and how such structures might propagate topological waves within and external to domains. For example, do chromatin modifiers exist that can stretch across from one domain to the next? Is it possible to trap domains using phased nucleosomes? The potential to examine a problem that is currently intractable is high.
Article Source: PNAS Website
Dividing a supercoiled DNA molecule into two independent topological domains
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.
PNAS Dec. 13, 2011; 108(50) 19973-19978
Published online before print November 28, 2011, doi: 10.1073/pnas.1109854108