Hin-mediated DNA knotting and recombining promote replicon dysfunction and mutation

<p>Abstract</p> <p>Background</p> <p>The genetic code imposes a dilemma for cells. The DNA must be long enough to encode for the complexity of an organism, yet thin and flexible enough to fit within the cell. The combination of these properties greatly favors DNA collisions, which can knot and drive recombination of the DNA. Despite the well-accepted propensity of cellular DNA to collide and react with itself, it has not been established what the physiological consequences are.</p> <p>Results</p> <p>Here we analyze the effects of recombined and knotted plasmids in <it>E. coli </it>using the Hin site-specific recombination system. We show that Hin-mediated DNA knotting and recombination (i) promote replicon loss by blocking DNA replication; (ii) block gene transcription; and (iii) cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid.</p> <p>Conclusion</p> <p>These results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.</p

The why and how of DNA unlinking

The nucleotide sequence of DNA is the repository of hereditary information. Yet, it is now clear that the DNA itself plays an active role in regulating the ability of the cell to extract its information. Basic biological processes, including control of gene transcription, faithful DNA replication and segregation, maintenance of the genome and cellular differentiation are subject to the conformational and topological properties of DNA in addition to the regulation imparted by the sequence itself. How do these DNA features manifest such striking effects and how does the cell regulate them? In this review, we describe how misregulation of DNA topology can lead to cellular dysfunction. We then address how cells prevent these topological problems. We close with a discussion on recent theoretical advances indicating that the topological problems, themselves, can provide the cues necessary for their resolution by type-2 topoisomerases

Topoisomerase II: a fitted mechanism for the chromatin landscape

The mechanism by which type-2A topoisomerases transport one DNA duplex through a transient double-strand break produced in another exhibits fascinating traits. One of them is the fine coupling between inter-domainal movements and ATP usage; another is their preference to transport DNA in particular directions. These capabilities have been inferred from in vitro studies but we ignore their significance inside the cell, where DNA configurations markedly differ from those of DNA in free solution. The eukaryotic type-2A enzyme, topoisomerase II, is the second most abundant chromatin protein after histones and its biological roles include the decatenation of newly replicated DNA and the relaxation of polymerase-driven supercoils. Yet, topoisomerase II is also implicated in other cellular processes such as chromatin folding and gene expression, in which the topological transformations catalysed by the enzyme are uncertain. Here, some capabilities of topoisomerase II that might be relevant to infer the enzyme performance in the context of chromatin architecture are discussed. Some aspects addressed are the importance of the DNA rejoining step to ensure genome stability, the regulation of the enzyme activity and of its putative structural role, and the selectively of DNA transport in the chromatin milieu

Using DNA supercoiling to control DNA minicircle shape

Non UBCUnreviewedAuthor affiliation: Baylor College of MedicineFacult

Diffusion of supercoiled DNA and the effect of base-flipping by Brownian dynamics(Knots and soft-matter physics: Topology of polymers and related topics in physics, mathematics and biology)

この論文は国立情報学研究所の電子図書館事業により電子化されました。Supercoiled DNAは、2本の環状鎖が絡み合っているlinkとみなせる。その絡み目数(linking number L_k)は、熱揺らぎのもとで保存する。我々ははしご型のモデルを作り、絡み目数と拡散の関係を調べた。はしごを何度かねじった後に右端と左端のビーズをFENEバネでつなぐことにより、絡み目数を保存する。その結果、拡散定数は絡み目数の線形関数であることが分かった。さらに我々はbase-flippingの拡散に与える影響を調べるため、FENEバネでつながれたペアの1つを切り離した。その結果拡散は、base-flippingを考慮しないモデルに比較して遅くなることが分かった。この傾向は角度ポテンシャルを考慮する時、より顕著となった。We have evaluated the diffusion constant of a ladder-like model of supercoiled DNA (see figure) in solution through Brownian dynamics with both hydrodynamic and excluded volume effects. After twisting the ladder we connect the ends so that its linking number L_k is conserved. We found that the diffusion constant is a linear function of L_k. In order to study the effect of base-flipping we disconnect the FENE spring potential that connects one of the pairs. The diffusion constant of the model with base-flipping becomes smaller especially when we take into account the angle potential

Bullied no more : When DNA shoves proteins around(Knots and soft-matter physics: Topology of polymers and related topics in physics, mathematics and biology)

この論文は国立情報学研究所の電子図書館事業により電子化されました。Most studies of protein-DNA interactions take a protein-centric perspective-giant proteins "bully" a static DNA polymer into a recognizable configuration. The structure of the protein is considered the primary determinant in the interaction, and DNA is considered, by comparison, merely a passive substrate. There are likely several reasons for this view, but the most important reason, perhaps, is that static crystal structures, which are the most vivid and compelling pictures we have, contain only a short fragment of DNA. The mechanistic explanations for protein-DNA recognition, therefore, usually arise from the structure of the protein. But protein structure does not tell the whole story. We propose that to understand protein-DNA interactions, a more holistic perspective must be taken. Protein-DNA interactions involve not just the protein, but also what we now know are incredibly dynamic DNA molecules, and the equally dynamic solvent molecules and counterions that surround them. Here we consider the ways that DNA topology can affect protein-DNA interactions, and focus, in particular, on the local, sequence-specific properties of DNA that do not occur when DNA is in the relaxed B-form as it is found in nearly all DNA crystal structures and is employed in the overwhelming majority of biophysical and biochemical studies of DNA structure and protein-DNA binding. DNA in cells is not inert like the linear B-form used in such experiments and it does not have naked ends. Instead, DNA in cells has topology, and topology affects: curvature, twist, kinking, base flipping, denaturation, and counterion concentrations, in addition to the likelihood that two DNA helices come together to form DNA juxtapositions

Contributions of the Combined Effects of Topoisomerase Mutations toward Fluoroquinolone Resistance in Escherichia coli▿

In defined, isogenic strains, at least three mutations, two of which must be in gyrA, were required to exceed the CLSI breakpoint for fluoroquinolone resistance. Strains with double mutations in both gyrA and parC had even higher MICs of fluoroquinolones than strains with totals of three mutations

Topoisomerase IV, alone, unknots DNA in E. coli

Knotted DNA has potentially devastating effects on cells. By using two site-specific recombination systems, we tied all biologically significant simple DNA knots in Escherichia coli. When topoisomerase IV activity was blocked, either with a drug or in a temperature-sensitive mutant, the knotted recombination intermediates accumulated whether or not gyrase was active. In contrast to its decatenation activity, which is strongly affected by DNA supercoiling, topoisomerase IV unknotted DNA independently of supercoiling. This differential supercoiling effect held true regardless of the relative sizes of the catenanes and knots. Finally, topoisomerase IV unknotted DNA equally well when DNA replication was blocked with hydroxyurea. We conclude that topoisomerase IV, not gyrase, unknots DNA and that it is able to access DNA in the cell freely. With these results, it is now possible to assign completely the topological roles of the topoisomerases in E. coli. It is clear that the topoisomerases in the cell have distinct and nonoverlapping roles. Consequently, our results suggest limitations in assigning a physiological function to a protein based upon sequence similarity or even upon in vitro biochemical activity