Positive Supercoiling Research Articles

Association of Sedentary Behavior with the Expression Levels of Biomarkers in Colorectal Cancer: Clinical Analysis of 228 Patients

There is an association between sedentary behavior and the risk of colorectal cancer (CRC), but the underlying mechanism is unclear. CRC is characterized by the changes in the expression levels of biomarkers, including voltage-gated proton channel Hv1, matrix-remodeling associated 5 (MXRA5), DEK (inducing positive supercoils into circular DNA) and protein inhibitor of activated signal transducer and activators of transcription 3 (PIAS3). Thus, sedentary behavior may affect the expression levels of these biomarkers in the colorectal tissue. Here, we recruited 228 CRC patients (128 males, 57.8 ± 7.8 years; 100 females, 57.7 ± 7.5 years) and 80 healthy subjects (48 males, 57.5 ± 6.8 years; 32 females, 56.9 ± 6.5 years) from March 7th, 2010 to May 6th, 2012. All the subjects were unrelated Han Chinese with the similar cultural and economic background. All the subjects were interviewed concerning sedentary time (sitting time categories: less than 1, 1-3, 4-6, and more than 6 h/day). The daily sedentary time of most CRC patients was more than 4 h/day, while the sedentary time of most healthy subjects was less than 3 h/day. The expression levels of Hv1, MXRA5 and DEK mRNAs and proteins were higher in CRC tissues and the levels of PIAS3 mRNA and protein were lower when the daily sedentary time was longer in CRC patients (p < 0.05). The daily sedentary time was correlated with the protein levels of CRC biomarkers. Furthermore, the sedentary time was positively related with body mass index but not daily calorie intake.

The centromeric nucleosome-like CENP–T–W–S–X complex induces positive supercoils into DNA

The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP–A. In addition to CENP–A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP–T–W–S–X complex. However, it is unclear how the CENP–T–W–S–X complex associates with centromere chromatin. Here, we demonstrate that the CENP–T–W–S–X complex binds preferentially to ∼100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP–T–W–S–X complex primarily binds to DNA as a (CENP–T–W–S–X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP–T–W–S–X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP–T or CENP–W, but not CENP–S or CENP–X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP–T–W–S–X complex. In summary, our work reveals the structural features and properties of the CENP–T–W–S–X complex for its localization to centromeres.

Regulation of Sister Chromosome Cohesion by the Replication Fork Tracking Protein SeqA

Analogously to chromosome cohesion in eukaryotes, newly replicated DNA in E. coli is held together by inter-sister linkages before partitioning into daughter nucleoids. In both cases, initial joining is apparently mediated by DNA catenation, in which replication-induced positive supercoils diffuse behind the fork, causing newly replicated duplexes to twist around each other. Type-II topoisomerase-catalyzed sister separation is delayed by the well-characterized cohesin complex in eukaryotes, but cohesion control in E. coli is not currently understood. We report that the abundant fork tracking protein SeqA is a strong positive regulator of cohesion, and is responsible for markedly prolonged cohesion observed at “snap” loci. Epistasis analysis suggests that SeqA stabilizes cohesion by antagonizing Topo IV-mediated sister resolution, and possibly also by a direct bridging mechanism. We show that variable cohesion observed along the E. coli chromosome is caused by differential SeqA binding, with oriC and snap loci binding disproportionally more SeqA. We propose that SeqA binding results in loose inter-duplex junctions that are resistant to Topo IV cleavage. Lastly, reducing cohesion by genetic manipulation of Topo IV or SeqA resulted in dramatically slowed sister locus separation and poor nucleoid partitioning, indicating that cohesion has a prominent role in chromosome segregation.

Differential contributions of the latch in Thermotoga maritima reverse gyrase to the binding of single-stranded DNA before and after ATP hydrolysis

Reverse gyrase catalyzes the ATP-dependent introduction of positive supercoils into DNA. Supercoiling requires the functional cooperation of its N-terminal helicase domain with the C-terminal topoisomerase domain. The helicase domain contains a superfamily 2 helicase core formed by two RecA domains, H1 and H2. We show here that a helicase domain lacking the latch, an insertion in H2, fails to close the cleft in the helicase core in response to nucleotide and DNA binding at the beginning of the catalytic cycle. In the presence of the pre-hydrolysis ATP analog ADP·BeFx, however, the closed conformer can still be formed in the absence of the latch. The helicase domain lacking the latch exhibits reduced DNA affinities. The energetic difference between the two nucleotide states involved in duplex separation is diminished, rationalizing the unwinding deficiency of reverse gyrase lacking the latch. The latch most strongly contributes to binding of single-stranded DNA in the post-hydrolysis state, before phosphate release. Our results are in line with contributions of the latch in determining the direction of strand passage, and in orienting the cleaved single-stranded DNA for re-ligation. At the same time, the latch may coordinate the re-ligation reaction with strand passage and with the nucleotide cycle.

Distinct Regions of the Escherichia coli ParC C-Terminal Domain Are Required for Substrate Discrimination by Topoisomerase IV

Type IIA DNA topoisomerases are essential enzymes that use ATP to maintain chromosome supercoiling and remove links between sister chromosomes. In Escherichia coli, the type IIA topoisomerase topo IV rapidly removes positive supercoils and catenanes from DNA but is significantly slower when confronted with negatively supercoiled substrates. The ability of topo IV to discriminate between positively and negatively supercoiled DNA requires the C-terminal domain (CTD) of one of its two subunits, ParC. To determine how the ParC CTD might assist with substrate discrimination, we identified potential DNA interacting residues on the surface of the CTD, mutated these residues, and tested their effect on both topo IV enzymatic activity and DNA binding by the isolated domain. Surprisingly, different regions of the ParC CTD do not bind DNA equivalently, nor contribute equally to the action of topo IV on different types of DNA substrates. Moreover, we find that the CTD contains an autorepressive element that inhibits activity on negatively supercoiled and catenated substrates, as well as a distinct region that aids in bending the DNA duplex that tracks through the enzyme's nucleolytic center. Our data demonstrate that the CTD is essential for proper engagement of both gate and transfer segment DNAs, reconciling different models to explain how topo IV discriminates between distinct DNAs topologies.

The Tail That Wags the Dog: Topoisomerase IV ParC C-Terminal Domain Controls Strand Passage Activity through Multipartite Topology-Dependent Interactions with DNA

The Tail That Wags the Dog: Topoisomerase IV ParC C-Terminal Domain Controls Strand Passage Activity through Multipartite Topology-Dependent Interactions with DNA

Insights from simulations into the mechanism of human topoisomerase I: Explanation for a seeming controversy in experiments

Human topoisomerase-I is a vital enzyme involved in cellular regulation of DNA supercoiling. We extend our previous work on wild type enzyme [13] to study how different enzyme mutants with various parts of the protein clamped by disulfide mutations affect DNA rotation. Three different mutants have been simulated; they are clamped enzyme-DNA systems in which the disulfide bridge is formed by replacing His367 and Ala499, Gly365 and Ser534, and, respectively, Leu429 and Lys436 with Cys pairs. The first of these mutants, a ‘distally clamped’ enzyme, mimics the experimental study of Carey et al. [11], which reports DNA rotation within the clamped enzyme. The second one, a ‘proximal clamp’, mimics the study of Woo et al. [12], who do not observe DNA rotation. The third is a newly suggested mutant that clamps the hinge for protein opening; we use it to test a hypothesis on negative supercoil relaxation. Our simulations show that the helical domain α5 totally melts in relaxation of positive supercoils when the enzyme is proximally clamped, while it preserves its structure very well within the distally clamped one. Moreover, a distally clamped protein permits DNA rotations in both directions, while the proximal clamp allows rotations only for negatively supercoiled DNA. These observations reconcile the two seemingly contradictory experimental findings, suggesting that subtle changes in the location of the disulfide bridge alter the mechanism significantly.

Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase IIα

Human topoisomerase IIα unlinks catenated chromosomes and preferentially relaxes positive supercoils. Supercoil chirality, twist density, and tension determine topoisomerase IIα relaxation rate and processivity. Strand passage rate is determined by the efficiency of transfer segment capture that is modulated by the topoisomerase C-terminal domains. Single-molecule measurements reveal the mechanism of chiral discrimination and tension dependence of supercoil relaxation by human topoisomerase IIα. Type IIA topoisomerases (Topo IIA) are essential enzymes that relax DNA supercoils and remove links joining replicated chromosomes. Human topoisomerase IIα (htopo IIα), one of two human isoforms, preferentially relaxes positive supercoils, a feature shared with Escherichia coli topoisomerase IV (Topo IV). The mechanistic basis of this chiral discrimination remains unresolved. To address this important issue, we measured the relaxation of individual supercoiled and "braided" DNA molecules by htopo IIα using a magnetic tweezers-based single-molecule assay. Our study confirmed the chiral discrimination activity of htopo IIα and revealed that the strand passage rate depends on DNA twist, tension on the DNA, and the C-terminal domain (CTD). Similar to Topo IV, chiral discrimination by htopo IIα results from chiral interactions of the CTDs with DNA writhe. In contrast to Topo IV, however, these interactions lead to chiral differences in relaxation rate rather than processivity. Increasing tension or twist disrupts the CTD-DNA interactions with a subsequent loss of chiral discrimination. Together, these results suggest that transfer segment (T-segment) capture is the rate-limiting step in the strand passage cycle. We propose a model for T-segment capture that provides a mechanistic basis for chiral discrimination and provides a coherent explanation for the effects of DNA twist and tension on eukaryotic type IIA topoisomerases.

Topological similarity between the 2μm plasmid partitioning locus and the budding yeast centromere: evidence for a common evolutionary origin?

The partitioning locus STB of the selfish plasmid, the 2μm circle, of Saccharomyces cerevisiae is essential for the propagation of this multi-copy extra-chromosomal DNA element with nearly chromosome-like stability. The functional competence of STB requires the plasmid-coded partitioning proteins Rep1 and Rep2 as well as host-coded proteins. Host factors that associate with STB in a Rep1- and Rep2-dependent manner also interact with centromeres, and play important roles in chromosome segregation. They include the cohesin complex and the centromere-specific histone H3 variant Cse4. The genetically defined point centromere of S. cerevisiae differs starkly from the much more widespread epigenetically specified regional centromeres of eukaryotes. The particularly small size of the S. cerevisiae centromere and the association of chromosome segregation factors with STB raise the possibility of an evolutionary link between these two partitioning loci. The unusual positive supercoiling harboured by the S. cerevisiae centromere and STB invivo in their functional states, unveiled by recent experiments, bolsters the notion of their potential descent from an ancestral plasmid partitioning locus.

Mechanical Stability of Mononucleosome Revealed by Optical Torque Wrench

The fundamental chromatin packing unit in eukaryotes is the nucleosome, where ∼147 base pairs of DNA are wrapped in ∼1.7 turns around a core histone octamer. A crucial question in biology is to explain how proteins are able to access DNA which is tightly bound in chromatin. For example, RNA polymerase must navigate through the nucleosome while transcribing DNA. Hence, the DNA-histone interactions play a key role in gene regulation. Single-molecule force spectroscopy is a powerful tool to probe this system. Prior studies have exerted linear tension to stretch both chromatin fibers and mononucleosome molecules, which have given information on the nature of the free-energy barrier for a particular disruption pathway. We develop a theoretical model including torsional constraints, which suggests that the disruption pathway may be strongly sensitive to the torsional loading of the nucleosome. This is of interest because helicases, polymerases, or other motor proteins may use a combination of force and torque to disrupt chromatin. Experimentally we apply force and torque simultaneously to disrupt a mononucleosome structure using an optical torque wrench. Positive supercoiling is found to destabilize the nucleosome while negative supercoiling has little effect, which is consistent with our model. By determining the influence of supercoiling density on the disruption barrier we obtain more detailed information about DNA-protein interaction strength in nucleosomes.

2P114 Direct observation of DNA positive supercoiling by reverse gyrase(04. Nucleic acid binding proteins,Poster)

2P114 Direct observation of DNA positive supercoiling by reverse gyrase(04. Nucleic acid binding proteins,Poster)

Crystal structures of Thermotoga maritima reverse gyrase: inferences for the mechanism of positive DNA supercoiling

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presented.

Reverse Gyrase Transiently Unwinds Double-Stranded DNA in an ATP-Dependent Reaction

Reverse gyrase is a unique DNA topoisomerase that catalyzes the introduction of positive supercoils into DNA in an ATP-dependent reaction. It consists of a helicase domain that functionally cooperates with a topoisomerase domain. Different models for the catalytic mechanism of reverse gyrase that predict a central role of the helicase domain have been put forward. The helicase domain acts as a nucleotide-dependent conformational switch that alternates between open and closed states with different affinities for single- and double-stranded DNA. It has been suggested that the helicase domain can unwind double-stranded regions, but helicase activity has not been demonstrated as yet. Here, we show that the isolated helicase domain and full-length reverse gyrase can transiently unwind double-stranded regions in an ATP-dependent reaction. The latch region of reverse gyrase, an insertion into the helicase domain, is required for DNA supercoiling. Strikingly, the helicase domain lacking the latch cannot unwind DNA, linking unwinding to DNA supercoiling. The unwinding activity may provide and stabilize the single-stranded regions required for strand passage by the topoisomerase domain, either de novo or by expanding already existing unpaired regions that may form at high temperatures.

Studying the interaction between gyrase and DNA using magnetic tweezers

The DNA gyrase of Escherichia coli plays an essential role in the life of this microorganism. It is unique among all topoisomerases because of its ability to introduce negative supercoils into DNA. This study investigated the single molecular interaction of E. coli gyrase with DNA using magnetic tweezers. The results showed that, in the absence of ATP, gyrase weakly binds the G and T segments. The stretched force of 0.7 pN can gradually destroy the binding, whereas that of 5.9 pN directly destroys it. Addition of high concentrations of norfloxacin enhances gyrase binding to both segments, making them adapt to 5.9 pN. DNA gyrase reduces the plectonemic dimension, which was determined by the bacterial enzyme and not by the pull force. Moreover, it has different affinities for positive supercoils, which it prefers, and negative supercoils. The time distribution of the dissociation of gyrase from DNA has a double-exponential form. We herein propose a model to explain this distribution and compare the results with those of other models.

Determining DNA supercoiling enthalpy by isothermal titration calorimetry

DNA supercoiling plays a critical role in certain essential DNA transactions, such as DNA replication, recombination, and transcription. For this reason, exploring energetics of DNA supercoiling is fundamentally important for understanding its biological functions. In this paper, using a unique property of DNA intercalators, such as ethidium bromide and daunorubicin, which bind to supercoiled, nicked, and relaxed DNA templates with different DNA-binding enthalpies, we determined DNA supercoiling enthalpy of plasmid pXXZ6, a 4.5 kb plasmid to be about 11.5 kcal/mol per linking number change. This determination allowed us to partition the DNA supercoiling free energy into enthalpic and entropic contributions where the unfavorable DNA supercoiling free energy exclusively originated from the large positive supercoiling enthalpy and was compensated by a large, favorable entropy term (TΔS).

Synergic and Opposing Activities of Thermophilic RecQ-like Helicase and Topoisomerase 3 Proteins in Holliday Junction Processing and Replication Fork Stabilization

RecQ family helicases and topoisomerase 3 enzymes form evolutionary conserved complexes that play essential functions in DNA replication, recombination, and repair, and in vitro, show coordinate activities on model recombination and replication intermediates. Malfunctioning of these complexes in humans is associated with genomic instability and cancer-prone syndromes. Although both RecQ-like and topoisomerase 3 enzymes are present in archaea, only a few of them have been studied, and no information about their functional interaction is available. We tested the combined activities of the RecQ-like helicase, Hel112, and the topoisomerase 3, SsTop3, from the thermophilic archaeon Sulfolobus solfataricus. Hel112 showed coordinate DNA unwinding and annealing activities, a feature shared by eukaryotic RecQ homologs, which resulted in processing of synthetic Holliday junctions and stabilization of model replication forks. SsTop3 catalyzed DNA relaxation and annealing. When assayed in combination, SsTop3 inhibited the Hel112 helicase activity on Holliday junctions and stimulated formation and stabilization of such structures. In contrast, Hel112 did not affect the SsTop3 DNA relaxation activity. RecQ-topoisomerase 3 complexes show structural similarity with the thermophile-specific enzyme reverse gyrase, which catalyzes positive supercoiling of DNA and was suggested to play a role in genome stability at high temperature. Despite such similarity and the high temperature of reaction, the SsTop3-Hel112 complex does not induce positive supercoiling and is thus likely to play different roles. We propose that the interplay between Hel112 and SsTop3 might regulate the equilibrium between recombination and anti-recombination activities at replication forks.

Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication

DNA topology plays a crucial role in all living cells. In prokaryotes, negative supercoiling is required to initiate replication and either negative or positive supercoiling assists decatenation. The role of DNA knots, however, remains a mystery. Knots are very harmful for cells if not removed efficiently, but DNA molecules become knotted in vivo. If knots are deleterious, why then does DNA become knotted? Here, we used classical genetics, high-resolution 2D agarose gel electrophoresis and atomic force microscopy to show that topoisomerase IV (Topo IV), one of the two type-II DNA topoisomerases in bacteria, is responsible for the knotting and unknotting of sister duplexes during DNA replication. We propose that when progression of the replication forks is impaired, sister duplexes become loosely intertwined. Under these conditions, Topo IV inadvertently makes the strand passages that lead to the formation of knots and removes them later on to allow their correct segregation.

DEK Regulates Hematopoietic Stem Engraftment and Progenitor Cell Proliferation

Abstract 1275Hematopoiesis is regulated by cell-cell and cytokine-cell interactions on hematopoietic stem (HSCs) and progenitor (HPCs) cells. In our continuing efforts to elucidate players involved in regulation of HSC and HPC growth, we focused on DEK, an abundant and unusual protein found in multicellular organisms. DEK has two DNA binding modules and has some affinity for specific DNA sequences, but primarily recognizes and binds to superhelical and cruciform DNA and induces positive supercoiling. DEK manifests multiple cellular activities, which include transcriptional repression and activation, mRNA processing, and chromatin architectural functions. We recently demonstrated that DEK modulates global heterochromatin integrity in vivo. Interestingly, DEK, can leave the cell and act as a chemoattractant for CD8+T cells and natural killer cells. Being intrigued that a nuclear protein was able to be secreted by hematopoietic cells, and act on other hematopoietic cells, we hypothesized that DEK might play a role in HSC/HPC function and hematopoiesis. In order to determine if DEK had an effect on steady state hematopoiesis, BM and spleen cells from DEK −/− mice were compared to that of wildtype (WT) control mice for absolute numbers and cycling status of HPC. Absolute numbers of CFU-GM, BFU-E, and CFU-GEMM per femur and per spleen were increased in DEK −/− mice. These effects were consistent with significantly increased percentages of HPCs in S-Phase of the cell cycle in DEK −/− BM and spleen, suggesting that DEK acts as a negative regulator of HPC proliferation in vivo. To confirm this, recombinant human DEK was tested for effects on HPC proliferation using unseparated mouse BM and low density human CB cells. DEK, dose-dependently suppressed colony formation by mouse BM CFU-GM stimulated by either IL-3 or GM-CSF, each alone; it did not influence colony formation stimulated by M-CSF alone. However, it dose-dependently inhibited CFU-GM colony formation by either IL-3, GM-CSF, or M-CSF when these cytokines were combined with the potent co-stimulating cytokine SCF. In fact, inhibition by DEK was greater on CFU-GM stimulated by the combination of IL-3, GM-CSF or M-CSF, each in the presence of SCF, compared to CFU-GM stimulated by IL-3, GM-CSF or M-CSF each alone in terms of percent inhibition, as well as the amount of DEK required to inhibit colony formation. Similar results were noted for HPCs present in human CB. This suggests that immature subsets of HPCs are more sensitive in vitro to the suppressive effects of DEK, than are the more mature HPCs. To determine if the DEK effects were directly or indirectly manifesting on the HPCs, DEK was assessed for effects on single isolated CD34+ cord blood cells, each in a single well stimulated by EPO, GM-CSF, IL-3, and SCF. DEK significantly decreased the number of wells containing a CFU-GM-, BFU-E-, or CFU-GEM- colony, demonstrating that DEK initiates it’s suppressive effect directly on HPC. Using a mouse competitive repopulating HSC assay in vivo, allows assessment of the short- and longer-term repopulating HSC, and transplantation of BM cells from primary to secondary lethally-irradiated recipients in a non-competitive assay describes the longer-term repopulating HSC, and can offer information on the self-renewal capacity of this population of HSCs. While there was no difference in the HSC repopulating capacity of DEK −/− and WT shorter-term repopulating cells (months 1 and 2 for blood chimerism), there was a significant decrease in DEK −/− compared to WT BM cell repopulation at month 4 in the blood, and month 6 in the BM. This decreased repopulation of DEK −/− compared to WT HSC was even more apparent in secondary mouse recipients suggesting that DEK played a positive role in engraftment of longer-term repopulating HSCs, and perhaps in the self-renewal capacity of mouse BM HSCs. These studies demonstrate a here-to-fore unknown role for DEK in the regulation of HPCs, HSCs and hematopoiesis. DEK could have separate effects on HPC and HSC, as suggested by the direct acting effects of DEK on single HPC. Alternatively, DEK may alone, or in addition allow HSC to favor a self-renewal, vs. a differentiation pathway to HPC. Thus, DEK has potent effects on HSCs, HPCs, and hematopoiesis, and may be of potential clinical value for enhancing HSC activity/proliferation in vivo, or in an ex-vivo situation. Disclosures:No relevant conflicts of interest to declare.

Conversion of Commensal Escherichia coli K-12 to an Invasive Form via Expression of a Mutant Histone-Like Protein

ABSTRACTThe HUαE38K, V42L mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397–16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUαE38K, V42L was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins BimEL and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUαE38K, V42L variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes.

Histone H3-variant Cse4-induced positive DNA supercoiling in the yeast plasmid has implications for a plasmid origin of a chromosome centromere

The Saccharomyces cerevisiae 2-μm plasmid is a multicopy selfish genome that resides in the nucleus. The genetic organization of the plasmid is optimized for stable, high-copy propagation in host-cell populations. The plasmid's partitioning system poaches host factors, including the centromere-specific histone H3-variant Cse4 and the cohesin complex, enabling replicated plasmid copies to segregate equally in a chromosome-coupled fashion. We have characterized the in vivo chromatin topology of the plasmid partitioning locus STB in its Cse4-associated and Cse4-nonassociated states. We find that the occupancy of Cse4 at STB induces positive DNA supercoiling, with a linking difference (ΔLk) contribution estimated between +1 and +2 units. One plausible explanation for this contrary topology is the presence of a specialized Cse4-containing nucleosome with a right-handed DNA writhe at a functional STB, contrasted by a standard histone H3-containing nucleosome with a left-handed DNA writhe at a nonfunctional STB. The similarities between STB and centromere in their nucleosome signature and DNA topology would be consistent with the potential origin of the unusual point centromere of budding yeast chromosomes from the partitioning locus of an ancestral plasmid.