Contributions of Sequence to the Higher-Order Structures of DNA.

One of the critical unanswered questions in genome biophysics is how the primary sequence of DNA bases influences the global properties of very-long-chain molecules. The local sequence-dependent features of DNA found in high-resolution structures introduce irregularities in the disposition of adjacent residues that facilitate the specific binding of proteins and modulate the global folding and interactions of double helices with hundreds of basepairs. These features also determine the positions of nucleosomes on DNA and the lengths of the interspersed DNA linkers. Like the patterns of basepair association within DNA, the arrangements of nucleosomes in chromatin modulate the properties of longer polymers. The intrachromosomal loops detected in genomic studies contain hundreds of nucleosomes, and given that the simulated configurations of chromatin depend on the lengths of linker DNA, the formation of these loops may reflect sequence-dependent information encoded within the positioning of the nucleosomes. With knowledge of the positions of nucleosomes on a given genome, methods are now at hand to estimate the looping propensities of chromatin in terms of the spacing of nucleosomes and to make a direct connection between the DNA base sequence and larger-scale chromatin folding.

What controls DNA looping?

The looping of DNA provides a means of communication between sequentially distant genomic sites that operate in tandem to express, copy, and repair the information encoded in the DNA base sequence. The short loops implicated in the expression of bacterial genes suggest that molecular factors other than the naturally stiff double helix are involved in bringing the interacting sites into close spatial proximity. New computational techniques that take direct account of the three-dimensional structures and fluctuations of protein and DNA allow us to examine the likely means of enhancing such communication. Here, we describe the application of these approaches to the looping of a 92 base-pair DNA segment between the headpieces of the tetrameric Escherichia coli Lac repressor protein. The distortions of the double helix induced by a second protein--the nonspecific nucleoid protein HU--increase the computed likelihood of looping by several orders of magnitude over that of DNA alone. Large-scale deformations of the repressor, sequence-dependent features in the DNA loop, and deformability of the DNA operators also enhance looping, although to lesser degrees. The correspondence between the predicted looping propensities and the ease of looping derived from gene-expression and single-molecule measurements lends credence to the derived structural picture.

Weak operator binding enhances simulated Lac repressor-mediated DNA looping.

The 50th anniversary of Biopolymers coincides closely with the like celebration of the discovery of the Escherichia coli (lac) lactose operon, a classic genetic system long used to illustrate the influence of biomolecular structure on function. The looping of DNA induced by the binding of the Lac repressor protein to sequentially distant operator sites on DNA continues to serve as a paradigm for understanding long-range genomic communication. Advances in analyses of DNA structures and in incorporation of proteins in computer simulations of DNA looping allow us to address long-standing questions about the role of protein-mediated DNA loop formation in transcriptional control. Here we report insights gained from studies of the sequence-dependent contributions of the natural lac operators to Lac repressor-mediated DNA looping. Novel superposition of the ensembles of protein-bound operator structures derived from NMR measurements reveals variations in DNA folding missed in conventional structural alignments. The changes in folding affect the predicted ease with which the repressor induces loop formation and the ways that DNA closes between the protein headpieces. The peeling of the auxiliary operators away from the repressor enhances the formation of loops with the 92-bp wildtype spacing and hints of a structural reason behind their weak binding.

Insights into Genome Architecture Deduced from the Properties of Short Lac Repressor-mediated DNA Loops.

Genomic DNA is vastly longer than the space allotted to it in a cell. The molecule must fold with a level of organization that satisfies the imposed spatial constraints as well as allows for the processing of genetic information. Key players in this organization include the negative supercoiling of DNA, which facilitates the unwinding of the double-helical molecule, and the associations of DNA with proteins, which partition the DNA into isolated loops, or domains. In order to gain insight into the principles of genome organization and to visualize the folding of spatially constrained DNA, we have developed new computational methods to identify the preferred three-dimensional pathways of protein-mediated DNA loops and to characterize the topological properties of these structures. Here we focus on the levels of supercoiling and the spatial arrangements of DNA in model nucleoprotein systems with two topological domains. We construct these systems by anchoring DNA loops in opposing orientations on a common protein-DNA assembly, namely the Lac repressor protein with two bound DNA operators. The linked pieces of DNA form a covalently closed circle such that the protein attaches to two widely spaced sites along the DNA. We examine the effects of operator spacing, loop orientation, and long-range contacts on overall chain configuration and topology and discuss our findings in the context of classic experiments on the effects of supercoiling and operator spacing on Lac repressor-mediated looping and recent work on the role of proteins as barriers that divide genomes into independent topological domains.