Requirements for DNA-Bridging Proteins to Act as Topological Barriers of the Bacterial Genome

Abstract

Bacterial genomes have been shown to be partitioned into several-kilobase-long chromosomal domains that are topologically independent from each other, meaning that change of DNA superhelicity in one domain does not propagate to neighbors. Both in vivo and in vitro experiments have been performed to question the nature of the topological barriers at play, leading to several predictions on possible molecular actors. Here, we address the question of topological barriers using polymer models of supercoiled DNA chains that are constrained such as to mimic the action of predicted molecular actors. More specifically, we determine under which conditions DNA-bridging proteins may act as topological barriers. To this end, we developed a coarse-grained bead-and-spring model and investigated its properties through Brownian dynamics simulations. As a result, we find that DNA-bridging proteins must exert rather strong constraints on their binding sites; they must block the diffusion of the excess of twist through the two binding sites on the DNA molecule and, simultaneously, prevent the rotation of one DNA segment relative to the other one. Importantly, not all DNA-bridging proteins satisfy this second condition. For example, single bridges formed by proteins that bind DNA nonspecifically, like H-NS dimers, are expected to fail with this respect. Our findings might also explain, in the case of specific DNA-bridging proteins like LacI, why multiple bridges are required to create stable independent topological domains. Strikingly, when the relative rotation of the DNA segments is not prevented, relaxation results in complex intrication of the two domains. Moreover, although the value of the torsional stress in each domain may vary, their differential is preserved. Our work also predicts that nucleoid-associated proteins known to wrap DNA must form higher protein-DNA complexes to efficiently work as topological barriers.

Impact of Self-Association on the Architectural Properties of Bacterial Nucleoid Proteins

“…For example, both DNA/macromolecules demixing and DNA supercoiling contribute to the compaction of the bacterial DNA, but the total compaction of the DNA coil is the sum of the two contributions only in a limited range of values of macromolecular concentration and superhelical density, whereas their interplay is much more complex outside from this range (22). In this respect, it will certainly be instructive in future work to use the models discussed in the present work to investigate the interplay of nucleoid proteins and macromolecular crowders (29,(52)(53)(54), transcription factors (55)(56)(57), or DNA supercoiling and topological insulators (23).…”

“…To conclude, we would like to mention that models similar to those discussed in the present work have recently been proposed to study the formation of the bacterial nucleoid through the demixing of DNA and non-binding globular macromolecules (51-54), the preferential localization of the nucleoid inside the cell ( 26), the mechanism of facilitated diffusion, by which proteins search for their targets along the DNA sequence (55-57), and the requirements for DNA-bridging proteins to act as topological barriers of the bacterial genome (23). All these models are compatible and it is possible to combine two (or more) of them to get a more complete and realistic description of bacterial cells (22).…”

“…Concentrations of nucleotides and proteins are of the same order of magnitude as in vivo ones. For DNA, each bead represents 7.5 base pairs (bp) and the chain contains 2880 beads, equivalent to 21600 bp, as in (20,22,23). Each protein chain contains 7 beads with index m (1 7 m ≤ ≤ ), where terminal beads 1 m = and 7 represent the two DNA-binding sites of each protein, whereas beads 2 m = and 6 (for Model I) or 2 m = , 3, 5 and 6 (for Model II) represent the isomerization sites of the protein (Fig.…”

“…Concentrations of nucleotides and proteins are of the same order of magnitude as in vivo ones. For DNA, each bead represents 7.5 base pairs (bp) and the chain contains 2880 beads, equivalent to 21600 bp, as in (20,22,23). Each protein chain contains 7 beads with index m (1…”

Bacterial transcription during growth arrest

“…Supercoils can diffuse along the length of the DNA until impeded by a topological barrier. Many protein–DNA interactions (including an RNAP elongation complex) can act as topological barriers to diffusion of superhelical density [ 45 ]. In fact, the lower-mobility and higher-density state of the nucleoid during growth arrest may also affect the distribution of superhelical density by impeding rotation of the DNA and bound proteins.…”