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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.

SUBMITTER: Joyeux M

PROVIDER: S-EPMC7420610 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Archaeal chromatin proteins Cren7 and Sul7d from Sulfolobus are DNA benders. To better understand their architectural roles in chromosomal DNA organization, we analyzed DNA compaction by Cren7 and Sis7d, a Sul7d family member, from Sulfolobus islandicus at the single-molecule (SM) level by total single-molecule internal reflection fluorescence microscopy (SM-TIRFM) and atomic force microscopy (AFM). We show that both Cren7 and Sis7d were able to compact singly tethered ? DNA into a highly condensed structure in a three-step process and that Cren7 was over an order of magnitude more efficient than Sis7d in DNA compaction. The two proteins were similar in DNA bending kinetics but different in DNA condensation patterns. At saturating concentrations, Sis7d formed randomly distributed clusters whereas Cren7 generated a single and highly condensed core on plasmid DNA. This observation is consistent with the greater ability of Cren7 than of Sis7d to bridge DNA. Our results offer significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaea.IMPORTANCE A long-standing question is how chromosomal DNA is packaged in Crenarchaeota, a major group of archaea, which synthesize large amounts of unique small DNA-binding proteins but in general contain no archaeal histones. In the present work, we tested our hypothesis that the two well-studied crenarchaeal chromatin proteins Cren7 and Sul7d compact DNA by both DNA bending and bridging. We show that the two proteins are capable of compacting DNA, albeit with different efficiencies and in different manners, at the single molecule level. We demonstrate for the first time that the two proteins, which have long been regarded as DNA binders and benders, are able to mediate DNA bridging, and this previously unknown property of the proteins allows DNA to be packaged into highly condensed structures. Therefore, our results provide significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaeota.

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

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