Cohesin Complex: Structure and Principles of Interaction with DNA.

Accurate duplication and separation of long linear genomic DNA molecules is associated with a number of purely mechanical problems. SMC complexes are key components of the cellular machinery that ensures decatenation of sister chromosomes and compaction of genomic DNA during division. Cohesin, one of the essential eukaryotic SMC complexes, has a typical ring structure with intersubunit pore through which DNA molecules can be threaded. Capacity of cohesin for such topological entrapment of DNA is crucial for the phenomenon of post-replicative association of sister chromatids better known as cohesion. Recently, it became apparent that cohesin and other SMC complexes are, in fact, motor proteins with a very peculiar movement pattern leading to formation of DNA loops. This specific process has been called loop extrusion. Extrusion underlies multiple functions of cohesin beyond cohesion, but molecular mechanism of the process remains a mystery. In this review, we summarized the data on molecular architecture of cohesin, effect of ATP hydrolysis cycle on this architecture, and known modes of cohesin-DNA interactions. Many of the seemingly disparate facts presented here will probably be incorporated in a unified mechanistic model of loop extrusion in the not-so-distant future.

Cohesin-Dependent Loop Extrusion: Molecular Mechanics and Role in Cell Physiology.

The most prominent representatives of multisubunit SMC complexes, cohesin and condensin, are best known as structural components of mitotic chromosomes. It turned out that these complexes, as well as their bacterial homologues, are molecular motors, the ATP-dependent movement of these complexes along DNA threads leads to the formation of DNA loops. In recent years, we have witnessed an avalanche-like accumulation of data on the process of SMC dependent DNA looping, also known as loop extrusion. This review briefly summarizes the current understanding of the place and role of cohesin-dependent extrusion in cell physiology and presents a number of models describing the potential molecular mechanism of extrusion in a most compelling way. We conclude the review with a discussion of how the capacity of cohesin to extrude DNA loops may be mechanistically linked to its involvement in sister chromatid cohesion.

Activity-driven chromatin organization during interphase: Compaction, segregation, and entanglement suppression.

In mammalian cells, the cohesin protein complex is believed to translocate along chromatin during interphase to form dynamic loops through a process called active loop extrusion. Chromosome conformation capture and imaging experiments have suggested that chromatin adopts a compact structure with limited interpenetration between chromosomes and between chromosomal sections. We developed a theory demonstrating that active loop extrusion causes the apparent fractal dimension of chromatin to cross-over between two and four at contour lengths on the order of 30 kilo-base pairs. The anomalously high fractal dimension [Formula: see text] is due to the inability of extruded loops to fully relax during active extrusion. Compaction on longer contour length scales extends within topologically associated domains (TADs), facilitating gene regulation by distal elements. Extrusion-induced compaction segregates TADs such that overlaps between TADs are reduced to less than 35% and increases the entanglement strand of chromatin by up to a factor of 50 to several Mega-base pairs. Furthermore, active loop extrusion couples cohesin motion to chromatin conformations formed by previously extruding cohesins and causes the mean square displacement of chromatin loci during lag times ([Formula: see text]) longer than tens of minutes to be proportional to [Formula: see text]. We validate our results with hybrid molecular dynamics-Monte Carlo simulations and show that our theory is consistent with experimental data. This work provides a theoretical basis for the compact organization of interphase chromatin, explaining the physical reason for TAD segregation and suppression of chromatin entanglements which contribute to efficient gene regulation.

Epigenetic marks uniquely tune the material properties of HP1α condensates.

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1α) with DNA contributes to the formation of condensed chromatin states. HP1α localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1α and dictating emergent functions of its condensates remains to be understood. Here, we leverage a reductionist system, composed of modified and unmodified histone H3 peptides, HP1α, and DNA, to examine the contribution of specific epigenetic marks to phase behavior of HP1α. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1α condensates, whereas peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1α phase separation. Using fluorescence microscopy and rheological approaches, we further demonstrate that H3K9me3 histone peptides modulate the dynamics and viscoelastic network properties of HP1α condensates in a concentration-dependent manner. Additionally, in cells exposed to uniaxial strain, we find there to be a decreased ratio of nuclear H3K9me3 to HP1α. These data suggest that HP1α-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells and in response to mechanostimulation.

Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

A cellulosomal double-dockerin module from Clostridium thermocellum shows distinct structural and cohesin-binding features.

Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components.

Phosphorylated HP1α-Nucleosome Interactions in Phase Separated Environments.

In the nucleus, transcriptionally silent genes are sequestered into heterochromatin compartments comprising nucleosomes decorated with histone H3 Lys9 trimethylation and a protein called HP1α. This protein can form liquid-liquid droplets in vitro and potentially organize heterochromatin through a phase separation mechanism that is promoted by phosphorylation. Elucidating the molecular interactions that drive HP1α phase separation and its consequences on nucleosome structure and dynamics has been challenging due to the viscous and heterogeneous nature of such assemblies. Here, we tackle this problem by a combination of solution and solid-state NMR spectroscopy, which allows us to dissect the interactions of phosphorylated HP1α with nucleosomes in the context of phase separation. Our experiments indicate that phosphorylated human HP1α does not cause any major rearrangements to the nucleosome core, in contrast to the yeast homologue Swi6. Instead, HP1α interacts specifically with the methylated H3 tails and slows the dynamics of the H4 tails. Our results shed light on how phosphorylated HP1α proteins may regulate the heterochromatin landscape, while our approach provides an atomic resolution view of a heterogeneous and dynamic biological system regulated by a complex network of interactions and post-translational modifications.

Reevaluation of bromodomain ligands targeting BAZ2A.

BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2-ICR, and the BAZ2/BRD9 cross-reactive GSK2801, interfere with the recognition of acetylated lysines in histones by the bromodomains of BAZ2A and of its BAZ2B paralog. The two chemical probes were tested in prostate cancer cell lines with opposite androgen susceptibility. BAZ2-ICR and GSK2801 showed different cellular efficacies in accordance with their unequal selectivity profiles. Concurrent inhibition of BAZ2 and BRD9 did not reproduce the effects observed with GSK2801, indicating possible off-targets for this chemical probe. On the other hand, the single BAZ2 inhibition by BAZ2-ICR did not phenocopy genetic ablation, demonstrating that bromodomain interference is not sufficient to strongly affect BAZ2A functionality and suggesting a PROTAC-based chemical ablation as an alternative optimization strategy and a possible therapeutic approach. In this context, we also present the crystallographic structures of BAZ2A in complex with the above chemical probes. Binding poses of TP-238 and GSK4027, chemical probes for the bromodomain subfamily I, and two ligands of the CBP/EP300 bromodomains identify additional headgroups for the development of BAZ2A ligands.

Conserved and divergent mechanisms of inner kinetochore assembly onto centromeric chromatin.

Kinetochores are large protein complexes built on centromeric chromatin that mediate chromosome segregation. The inner kinetochore, or constitutive centromere-associated network (CCAN), assembles onto centromeres defined by centromere protein A (CENP-A) nucleosomes (CENP-ANuc), and acts as a platform for the regulated assembly of the microtubule-binding outer kinetochore. Recent cryo-EM work revealed structural conservation of CCAN, from the repeating human regional centromeres to the point centromere of budding yeast. Centromere recognition is determined mainly through engagement of duplex DNA proximal to the CENP-A nucleosome by a DNA-binding CENP-LN channel located at the core of CCAN. Additional DNA interactions formed by other CCAN modules create an enclosed DNA-binding chamber. This configuration explains how kinetochores maintain their tight grip on centromeric DNA to withstand the forces of chromosome segregation. Defining the higher-order architecture of complete kinetochore assemblies with implications for understanding the 3D organisation of regional centromeres and mechanisms of kinetochore dynamics, including how kinetochores sense and respond to tension, are important future directions.

FREEDA: An automated computational pipeline guides experimental testing of protein innovation.

Cell biologists typically focus on conserved regions of a protein, overlooking innovations that can shape its function over evolutionary time. Computational analyses can reveal potential innovations by detecting statistical signatures of positive selection that lead to rapid accumulation of beneficial mutations. However, these approaches are not easily accessible to non-specialists, limiting their use in cell biology. Here, we present an automated computational pipeline FREEDA that provides a simple graphical user interface requiring only a gene name; integrates widely used molecular evolution tools to detect positive selection in rodents, primates, carnivores, birds, and flies; and maps results onto protein structures predicted by AlphaFold. Applying FREEDA to >100 centromere proteins, we find statistical evidence of positive selection within loops and turns of ancient domains, suggesting innovation of essential functions. As a proof-of-principle experiment, we show innovation in centromere binding of mouse CENP-O. Overall, we provide an accessible computational tool to guide cell biology research and apply it to experimentally demonstrate functional innovation.

Evidence for low nanocompaction of heterochromatin in living embryonic stem cells.

Despite advances in the identification of chromatin regulators and genome interactions, the principles of higher-order chromatin structure have remained elusive. Here, we applied FLIM-FRET microscopy to analyse, in living cells, the spatial organisation of nanometre range proximity between nucleosomes, which we called "nanocompaction." Both in naive embryonic stem cells (ESCs) and in ESC-derived epiblast-like cells (EpiLCs), we find that, contrary to expectations, constitutive heterochromatin is much less compacted than bulk chromatin. The opposite was observed in fixed cells. HP1α knockdown increased nanocompaction in living ESCs, but this was overridden by loss of HP1ß, indicating the existence of a dynamic HP1-dependent low compaction state in pluripotent cells. Depletion of H4K20me2/3 abrogated nanocompaction, while increased H4K20me3 levels accompanied the nuclear reorganisation during EpiLCs induction. Finally, the knockout of the nuclear cellular-proliferation marker Ki-67 strongly reduced both interphase and mitotic heterochromatin nanocompaction in ESCs. Our data indicate that, contrary to prevailing models, heterochromatin is not highly compacted at the nanoscale but resides in a dynamic low nanocompaction state that depends on H4K20me2/3, the balance between HP1 isoforms, and Ki-67.

Analysis of absolute amounts of two meiotic cohesin subunits, RAD21L and REC8, in mouse spermatocytes.

RAD2lL and REC8, meiosis-specific paralogs of the canonical cohesin subunit RAD21, are essential for proper formation of axial/lateral elements of the synaptonemal complex, synapsis of homologous chromosomes, and crossover recombination in mammalian meiosis. However, how many meiotic cohesins are present in germ cells has not been investigated because of the lack of an appropriate method of analysis. In the present study, to examine the intracellular amount of meiotic cohesins, we generated two strains of knock-in (KI) mice that expressed a 3×FLAG-tag at the C-terminus of RAD21L or REC8 protein using the CRISPR/Cas9 genome editing system. Both KI mice were fertile. Western blot analyses and immunocytochemical studies revealed that expression levels and localization patterns of both RAD21L-3×FLAG and REC8-3×FLAG in KI mice were similar to those in wild-type mice. After confirming that tagging of endogenous RAD21L and REC8 with 3×FLAG did not affect their expression profiles, we evaluated the levels of RAD21L-3×FLAG and REC8-3×FLAG in the testes of 2-week-old mice in which only RAD21L and REC8 but little RAD21 are expressed in the meiocytes. By comparing the band intensities of testicular RAD21L-3×FLAG and REC8-3×FLAG with 3×FLAG-tagged recombinant proteins of known concentrations in western blot analysis, we found that there were approximately 413,000 RAD21L and 453,000 REC8 molecules per spermatocyte in the early stages of prophase I. These findings provide new insights into the role played by cohesins in the process of meiotic chromosome organization in mammalian germ cells.

Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies.

The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.

RUP2 facilitates UVR8 redimerization via two interfaces.

The plant UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) exists as a homodimer in its inactive ground state. Upon UV-B exposure, UVR8 monomerizes and interacts with a downstream key regulator, the CONSTITUTIVE PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA (COP1/SPA) E3 ubiquitin ligase complex, to initiate UV-B signaling. Two WD40 proteins, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 directly interact with monomeric UVR8 and facilitate UVR8 ground state reversion, completing the UVR8 photocycle. Here, we reconstituted the RUP-mediated UVR8 redimerization process in vitro and reported the structure of the RUP2-UVR8W285A complex (2.0 Å). RUP2 and UVR8W285A formed a heterodimer via two distinct interfaces, designated Interface 1 and 2. The previously characterized Interface 1 is found between the RUP2 WD40 domain and the UVR8 C27 subregion. The newly identified Interface 2 is formed through interactions between the RUP2 WD40 domain and the UVR8 core domain. Disruption of Interface 2 impaired UV-B induced photomorphogenic development in Arabidopsis thaliana. Further biochemical analysis indicated that both interfaces are important for RUP2-UVR8 interactions and RUP2-mediated facilitation of UVR8 redimerization. Our findings suggest that the two-interface-interaction mode is adopted by both RUP2 and COP1 when they interact with UVR8, marking a step forward in understanding the molecular basis that underpins the interactions between UVR8 and its photocycle regulators.

Multifaceted roles of YEATS domain-containing proteins and novel links to neurological diseases.

The so-called Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain-containing proteins, hereafter referred to as YD proteins, take control over the transcription by multiple steps of regulation either involving epigenetic remodelling of chromatin or guiding the processivity of RNA polymerase II to facilitate elongation-coupled mRNA 3' processing. Interestingly, an increasing amount of evidence suggest a wider repertoire of YD protein's functions spanning from non-coding RNA regulation, RNA-binding proteins networking, post-translational regulation of a few signalling transduction proteins and the spindle pole formation. However, such a large set of non-canonical roles is still poorly characterized. Notably, four paralogous of human YEATS domain family members, namely eleven-nineteen-leukaemia (ENL), ALL1-fused gene from chromosome 9 protein (AF9), YEATS2 and glioma amplified sequence 41 (GAS41), have a strong link to cancer yet new findings also highlight a potential novel role in neurological diseases. Here, in an attempt to more comprehensively understand the complexity of four YD proteins and to gain more insight into the novel functions they may accomplish in the neurons, we summarized the YD protein's networks, systematically searched and reviewed the YD genetic variants associated with neurodevelopmental disorders and finally interrogated the model organism Drosophila melanogaster.

Assembly and interaction of core subunits of BAF complexes and crystal study of the SMARCC1/SMARCE1 binary complex.

The multi-subunit ATP-dependent chromatin remodeling factor SWI/SNF complex is a fundamental regulator of gene transcription. The SWI/SNF complex in mammals, also called the BAF complex, consists of 9-12 subunits. Genomic functional studies have found that 20%-25% of human cancers are caused by mutations in genes encoding this complex. For the assembly of the BAF complex, BAF47 (SMARCB1), BAF57 (SMARCE1), BAF155 (SMARCC1)/BAF170 (SMARCC2), and BAF60 A/B/C (SMARCD1/2/3) form a core complex. However, the assembly mechanism of the BAF core subunit remains unclear. In this study, the assembly mechanism and structure of this complex and the interactions between its subunits were investigated. We co-expressed SMARCC1(447-966)/SMARCD1(129-471), SMARCC1(447-966)/SMARCE1(210-284) and SMARCC1(862-966)/SMARCE1(210-284) binary complex, SMARCC1(862-966)/SMARCD1(129-471)/SMARCE1(210-284) ternary complex SMARCC1(353-966)/SAMRCD1(129-471)/SMARCB1(110-385)/SAMRCE1(210-284) tetrameric complexes, and obtained crystals of the SMARCC1(862-966)/SMARCE1(210-284) and SMARCC1(883-966)/SMARCE1(210-284) binary complex，and the SMARCC1(883-966)/SMARCE1(210-284) crystal received a set of diffraction data of 3.2 Å. Our experimental results demonstrate the assembly mechanism between the core subunit quaternary complexes of the BAF complex and the interacting amino acid fragment regions and the SMARCC1/SMARCE1 optimal amino acid fragment binary complex crystals. Our study provides a theoretical basis for the development of cancer and related drug research based on protein structure.

Dynamics and mechanism of dimer dissociation of photoreceptor UVR8.

Photoreceptors are a class of light-sensing proteins with critical biological functions. UVR8 is the only identified UV photoreceptor in plants and its dimer dissociation upon UV sensing activates UV-protective processes. However, the dissociation mechanism is still poorly understood. Here, by integrating extensive mutations, ultrafast spectroscopy, and computational calculations, we find that the funneled excitation energy in the interfacial tryptophan (Trp) pyramid center drives a directional Trp-Trp charge separation in 80 ps and produces a critical transient Trp anion, enabling its ultrafast charge neutralization with a nearby positive arginine residue in 17 ps to destroy a key salt bridge. A domino effect is then triggered to unzip the strong interfacial interactions, which is facilitated through flooding the interface by channel and interfacial water molecules. These detailed dynamics reveal a unique molecular mechanism of UV-induced dimer monomerization.

Phosphorylation tunes elongation propensity and cohesiveness of INCENP's intrinsically disordered region.

The inner centromere protein, INCENP, is crucial for correct chromosome segregation during mitosis. It connects the kinase Aurora B to the inner centromere allowing this kinase to dynamically access its kinetochore targets. However, the function of its central, 440-residue long intrinsically disordered region (IDR) and its multiple phosphorylation sites is unclear. Here, we determined the conformational ensemble of INCENP's IDR, systematically varying the level of phosphorylation, using all-atom and coarse-grain molecular dynamics simulations. Our simulations show that phosphorylation expands INCENP's IDR, both locally and globally, mainly by increasing its overall net charge. The disordered region undergoes critical globule-to-coil conformational transitions and the transition temperature non-monotonically depends on the degree of phosphorylation, with a mildly phosphorylated case of neutral net charge featuring the highest collapse propensity. The IDR transitions from a multitude of globular states, accompanied by several specific internal contacts that reduce INCENP length by loop formation, to weakly interacting and highly extended coiled conformations. Phosphorylation critically shifts the population between these two regimes. It thereby influences cohesiveness and phase behavior of INCENP IDR assemblies, a feature presumably relevant for INCENP's function in the chromosomal passenger complex. Overall, we propose the disordered region of INCENP to act as a phosphorylation-regulated and length-variable component, within the previously defined "dog-leash" model, that thereby regulates how Aurora B reaches its targets for proper chromosome segregation.

Evolutionary mode for the functional preservation of fast-evolving Drosophila telomere capping proteins.

DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon we have investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on our results and on homology-based three-dimensional models for Ver and Moi, we conclude that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. We propose that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. We also provide here the first evidence on Moi DNA binding.

Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites.

The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active DNA loop extrusion. MukBEF activity within the replication terminus macrodomain is suppressed by the sequence-specific unloader MatP. Here, we present the complete atomic structure of MukBEF in complex with MatP and DNA as determined by electron cryomicroscopy (cryo-EM). The complex binds two distinct DNA double helices corresponding to the arms of a plectonemic loop. MatP-bound DNA threads through the MukBEF ring, while the second DNA is clamped by the kleisin MukF, MukE, and the MukB ATPase heads. Combinatorial cysteine cross-linking confirms this topology of DNA loop entrapment in vivo. Our findings illuminate how a class of near-ubiquitous DNA organizers with important roles in genome maintenance interacts with the bacterial chromosome.