Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis.

Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish "condensinopathies" as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size.

Higher Order Protein Catenation Leads to an Artificial Antibody with Enhanced Affinity and In Vivo Stability.

The chemical topology is a unique dimension for protein engineering, yet the topological diversity and architectural complexity of proteins remain largely untapped. Herein, we report the biosynthesis of complex topological proteins using a rationally engineered, cross-entwining peptide heterodimer motif derived from p53dim (an entangled homodimeric mutant of the tetramerization domain of the tumor suppressor protein p53). The incorporation of an electrostatic interaction at specific sites converts the p53dim homodimer motif into a pair of heterodimer motifs with high specificity for directing chain entanglement upon folding. Its combination with split-intein-mediated ligation and/or SpyTag/SpyCatcher chemistry facilitates the programmed synthesis of protein heterocatenane or [n]catenanes in cells, leading to a general and modular approach to complex protein catenanes containing various proteins of interest. Concatenation enhances not only the target protein's affinity but also the in vivo stability as shown by its prolonged circulation time in blood. As a proof of concept, artificial antibodies have been developed by embedding a human epidermal growth factor receptor 2-specific affibody onto the [n]catenane scaffolds and shown to exhibit a higher affinity and a better pharmacokinetic profile than the wild-type affibody. These results suggest that topology engineering holds great promise in the development of therapeutic proteins.

Identification of hemicatenane-specific binding proteins by fractionation of HeLa nuclei extracts.

DNA hemicatenanes (HCs) are four-way junctions in which one strand of a double-stranded helix is catenated with one strand of another double-stranded DNA. Frequently mentioned as DNA replication, recombination and repair intermediates, they have been proposed to participate in the spatial organization of chromosomes and in the regulation of gene expression. To explore potential roles of HCs in genome metabolism, we sought to purify proteins capable of binding specifically HCs by fractionating nuclear extracts from HeLa cells. This approach identified three RNA-binding proteins: the Tudor-staphylococcal nuclease domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing family, the paraspeckle protein component 1 and the splicing factor proline- and glutamine-rich protein. Since these proteins were partially pure after fractionation, truncated forms of these proteins were expressed in Escherichia coli and purified to near homogeneity. The specificity of their interaction with HCs was re-examined in vitro. The two truncated purified SND1 proteins exhibited specificity for HCs, opening the interesting possibility of a link between the basic transcription machinery and HC structures via SND1.

Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases.

Type IA DNA topoisomerases work with a unique mechanism of strand passage through an enzyme-bridged, ssDNA gate, thus enabling them to carry out diverse reactions in processing structures important for replication, recombination, and repair. Here we report a unique reaction mediated by an archaeal type IA topoisomerase, the synthesis and dissolution of hemicatenanes. We cloned, purified, and characterized an unusual type IA enzyme from a hyperthermophilic archaeum, Nanoarchaeum equitans, which is split into two pieces. The recombinant heterodimeric enzyme has the expected activities in its preference of relaxing negatively supercoiled DNA. Its amino acid sequence and cleavage site sequence analysis suggest that it is topoisomerase III, and therefore we named it "NeqTop3." At high enzyme concentrations, NeqTop3 can generate high-molecular-weight DNA networks. Biochemical and electron microscopic data indicate that the DNA networks are connected through hemicatenane linkages. The hemicatenane formation likely is mediated by the single-strand passage through denatured bubbles in the substrate DNA under high temperature. NeqTop3 at lower concentrations can reverse hemicatenanes. A complex of human topoisomerase 3α, Bloom helicase, and RecQ-mediated genome instability protein 1 and 2 can partially disentangle the hemicatenane network. Both the formation and dissolution of hemicatenanes by type IA topoisomerases demonstrate that these enzymes have an important role in regulating intermediates from replication, recombination, and repair.

Synthesis of Hemicatenanes for the Study of Type IA Topoisomerases.

Hemicatenane is a structure that forms when two DNA duplexes are physically linked through a single-stranded crossover. It is proposed to be an intermediate resulting from double Holliday junction (dHJ) dissolution, repair of replication stalled forks and late stage replication. Our previous study has shown that hemicatenane can be synthesized and dissolved in vitro by hyperthermophilic type IA topoisomerases. Here we present the protocol of hemicatenane synthesis and its structure detection by 2D agarose gel electrophoresis. The generated product can be used as a substrate to study the biochemical mechanism of hemicatenane processing reactions.

The Localization and Action of Topoisomerase IV in Escherichia coli Chromosome Segregation Is Coordinated by the SMC Complex, MukBEF.

The type II topoisomerase TopoIV, which has an essential role in Escherichia coli chromosome decatenation, interacts with MukBEF, an SMC (structural maintenance of chromosomes) complex that acts in chromosome segregation. We have characterized the intracellular dynamics of individual TopoIV molecules and the consequences of their interaction with MukBEF clusters by using photoactivated-localization microscopy. We show that ~15 TopoIV molecules per cell are associated with MukBEF clusters that are preferentially localized to the replication origin region (ori), close to the long axis of the cell. A replication-dependent increase in the fraction of immobile molecules, together with a proposed catalytic cycle of ~1.8 s, is consistent with the majority of active TopoIV molecules catalyzing decatenation, with a minority maintaining steady-state DNA supercoiling. Finally, we show that the MukB-ParC interaction is crucial for timely decatenation and segregation of newly replicated ori DNA.

Interlocked systems in nanomedicine.

The concept of Nanomedicine emerged along with the new millennium, and it is expected to provide solutions to some of modern medicine's unsolved problems. Nanomedicine offers new hopes in several critical areas such as cancer treatment, viral and bacterial infections, medical imaging, tissue regeneration, and theranostics. To explore all these applications, a wide variety of nanomaterials have been developed which include liposomes, dendrimers, nanohydrogels and polymeric, metallic and inorganic nanoparticles. Recently, interlocked systems, namely rotaxanes and catenanes, have been incorporated into some of these chemical platforms in an attempt to improve their performance. This review focus on the nanomedicine applications of nanomaterials containing interlocked structures. The introduction gives an overview on the significance of interdisciplinary science in the progress of the nanomedicine field, and it explains the evolution of interlocked molecules until their application in nanomedicine. The following sections are organized by the type of interlocked structure, and it comprises details of the in vitro and/or in vivo experiments involving each material: rotaxanes as imaging agents, rotaxanes as cytotoxic agents, rotaxanes as peptide transporters, mechanized silica nanoparticles as stimuli responsive drug delivery systems, and polyrotaxanes as drug and gene delivery systems.

Chemical peristalsis.

Molecules that emulate in part the remarkable capabilities of protein motors were recently chemically synthesized. A promising approach is based on physically interlocked macromolecular complexes such as rotaxanes and catenanes. Using the latter, Leigh et al. [Leigh, D. A., Wong, J. K. Y., Dehez, F. & Zerbetto, F. (2003) Nature 424, 174-179] constructed a molecular rotor in which two small rings are induced by pulses of light to move unidirectionally around a third, larger ring. The mechanism is similar to that by which a peristaltic pump operates. Unlike macroscopic peristalsis, however, in which a traveling wave forces material through a series of one-way valves, the chemical peristaltic mechanism does not directly cause the small rings to move but only alters the energetics, with the motion itself arising by thermal activation over energy barriers. Engines operating by this mechanism are "Brownian" motors. Here we describe a minimal two-state mechanism for a catenane-based molecular motor. Although fluctuations caused by equilibrium processes cannot drive directed motion, nonequilibrium fluctuations, whether generated externally or by a far-from-equilibrium chemical reaction, can drive rotation even against an external torque. We discuss a possible architecture for input and output of information and energy between the motor and its environment and give a simple expression for the maximum thermodynamic efficiency. The proposed Brownian motor mechanism is consistent with the high efficiency observed by Yasuda et al. [Yasuda, Y., Noji, H., Kinoshita, K. & Yoshida, M. (1998) Cell 93, 1117-1124] for the F(1)-ATP synthase operating as an ATP-powered molecular rotor.