Ring-shaped sliding clamps and clamp loader ATPases are essential factors for rapid and accurate DNA replication. The clamp ring is opened and resealed at the primer-template junctions by the ATP-fueled clamp loader function. The processivity of the DNA polymerase is conferred by its attachment to the clamp loaded onto the DNA. In eukarya and archaea, the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) play crucial roles as the clamp loader and the clamp, respectively. Here, we report the electron microscopic structure of an archaeal RFC-PCNA-DNA complex at 12-Å resolution. This complex exhibits excellent fitting of each atomic structure of RFC, PCNA, and the primed DNA. AAA ϩ ATPase ͉ clamp loader ͉ DNA replication ͉ electron microscopy ͉ single-particle analysis I n highly processive genomic DNA duplication, the DNA polymerase is tethered on the DNA strand through a direct interaction with the sliding clamp, which is topologically linked to the DNA by the action of the clamp loader (1). In this reaction, the clamp loader opens and reseals the clamp ring at the primer-template junctions in an ATP-dependent manner. Functional (2-8) and structural (9-12) analyses have indicated that the clamp-loading mechanism is conserved across the domains of life (13-15). All of the sliding clamps from phage to eukarya form similar planer rings, despite their distinct subunit compositions and lower sequence identities. Likewise, the clamp loader complexes from various organisms commonly exist as pentameric complexes with similar subunit configurations. The complexes have a unique oligomeric shape with the open ring in the N-terminal regions of each subunit, which folds into an architecture classified within the AAA ϩ ATPase superfamily (16), while the C-terminal regions form the closed ''collar'' structure. The crystal structure of the yeast clamp loader, replication factor C (RFC), in complex with the sliding clamp, proliferating cell nuclear antigen (PCNA), revealed their detailed contact mode and the elegant match of the spiral configuration of the Nterminal domains of RFC with that of the double-stranded (ds) DNA, and thus allowed the reasonable model building of the RFC-PCNA binary complex docked with a DNA duplex (12).We previously reported the 23-Å resolution EM structure of a clamp-loading RFC-PCNA-DNA ternary complex from Pyrococcus furiosus (Pfu), which was stabilized by introducing a nonhydrolyzable ATP analog, ATP␥S (17). The structure showed the two building blocks, a larger horseshoe and a smaller closed ring. It appeared the best interpretation based on the available data that the horseshoe and the closed ring correspond to RFC and PCNA, respectively. Although the atomic structures of the PCNA trimer (18) and RFC small subunits (RFCSs) (11) were available, along with the information about the 1:4 stoichiometry for RFC large subunit (RFCL) and RFCS in the RFC hetero-pentamer (5), the fitting of the atomic model into the EM map was not completely satisfactory, and some ambiguity remain...

No abstract

Intrinsically disordered (ID) regions of proteins are recognized to be involved in biological processes such as transcription, translation, and cellular signal transduction. Despite the important roles of ID regions, effective methods to observe these thin and flexible structures directly were not available. Herein, we use high-speed atomic force microscopy (AFM) to observe the heterodimeric FACT (facilitates chromatin transcription) protein, which is predicted to have large ID regions in each subunit. Successive AFM images of FACT on a mica surface, captured at rates of 5-17 frames per second, clearly reveal two distinct tail-like segments that protrude from the main body of FACT and fluctuate in position. Using deletion mutants of FACT, we identify these tail segments as the two major ID regions predicted from the amino acid sequences. Their mechanical properties estimated from the AFM images suggest that they have more relaxed structures than random coils. These observations demonstrate that this state-of-the-art microscopy method can be used to characterize unstructured protein segments that are difficult to visualize with other experimental techniques.

Inositol 1,4,5-trisphosphate receptor (IP 3 R) is a highly controlled calcium (Ca 2؉ ) channel gated by inositol 1,4,5-trisphosphate (IP 3 ). Multiple regulators modulate IP 3 -triggered pore opening by binding to discrete allosteric sites within IP 3 R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca 2؉ , the most pivotal regulator, induced marked structural changes in the tetrameric IP 3 R purified from mouse cerebella. Electron microscopy of the IP 3 R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca 2؉ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP 3 -binding domains, assigned by binding to heparin-gold. Ca 2؉ -dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca 2؉ appeared to regulate IP 3 gating activity through the rearrangement of functional domains.Inositol 1,4,5-trisphosphate receptor (IP 3 R) 1 is a tetrameric ion channel that release Ca 2ϩ from intracellular stores in response to the binding of 1,4,5-trisphosphate (IP 3 ), a second messenger generated by various extracellular stimuli, neurotransmitters, neuromodulators, hormones, and lights (1, 2). The IP 3 R is widely distributed in living systems and plays pivotal roles in fundamental processes including fertilization, cellular proliferation and differentiation, cellular signaling, and vesicle secretion (2). Molecular cloning studies have revealed that there are three isoforms of IP 3 R and that alternative splicing results in several variants of the IP 3 R (2). These divergent primary structures of the IP 3 R and their differential distributions have been assumed to award the functional diversity of IP 3 R by nature.The most characterized type 1 IP 3 R (IP 3 R1), a predominant type in rodent cerebellar endoplasmic reticulum (ER) and spine apparatus, plays an integral role in Ca 2ϩ signaling (3-5) and neural plasticity (6, 7). The protomer of IP 3 R1, a 2749-amino acid polypeptide (M r 313,000), contains the IP 3 -binding core (residues 226 -578), membrane-spanning domains (residues 2276 -2589), and widespread allosteric sites for intracellular effector molecules (Ca 2ϩ , calmodulin, and ATP) and for phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, Ca 2ϩ / calmodulin-dependent protein kinase II, and tyrosine kinase) (2). These cumulative allosteric regulations imply a structural paradigm for global conformational changes within the higher ordered structure of IP 3 R1.Because Ca 2ϩ rigorously determines the channel activity of IP 3 R and Ca 2ϩ -dependent behavior of IP 3 R is considered to be crucial for spatiotemporal organizations of Ca 2ϩ signaling (1, 4), the most important regulator for IP 3 R is Ca 2ϩ per se. Previous functional analysis indicates that a low Ca 2ϩ level acts a...

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.

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramerformsacentralporewithdimensionsof23Å indiameterand ϳ50 Å in depth. -Cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.Vibrio parahaemolyticus is a Gram-negative marine bacterium known to be one of the major causes of pandemic seafoodborne gastroenteritis. V. parahaemolyticus possesses two circular replicons of 3.2 and 1.9 megabase pairs, which might confer an advantage by enabling DNA replication in seawater of lower temperature and/or low nutritional value (1, 2). Such an advantage would potentially increase risks of food intoxication by allowing explosive expansion of the population of the microorganism. Hemolysis on Wagatsuma agar (a blood agar), known as the Kanagawa phenomenon, is associated with human pathogenic strains of V. parahaemolyticus. A major virulence factor of this pathogen is the thermostable direct hemolysin (TDH) 7 (3-5), which has a variety of biological activities including hemolytic activity, cardiotoxicity, and enterotoxicity. There are two copies of the tdh gene (or its homologue trh) in pathogenic V. parahaemolyticus, indicating the importance of this exotoxin for survival of the organism (2). The mature form of TDH consists of 165 amino acids, including a single intramolecular disulfide bond, but no close homologue of TDH has been found in other organisms. The significance of Arg 46 , Gly 62 , Trp 65 , and Gly 90 residues on hemolysis was determined by site-directed mutagenesis (3, 6).The common features of the bacterial pore-forming toxin are as follows. 1) It is released as a soluble monomer into the extra-bacterial space. 2) It oligomerizes to form a pore at the host cell membrane (7,8). An earlier study reported that TDH acted as a pore-forming toxin, creating a functional pore ϳ20 Å in diameter (reviewed in Ref. 3). We previously constructed a low resolution C 4 symmetric model of tetrameric TDH in solution based on small angle x-ray scattering (SAXS), transmission electron microscopy (TEM), and analytical ultracentrifugation (9). However, the precise structure and the mechanism for its pore-forming toxicity are still unknown. Several bacterial toxins, including TDH, show paradoxical responses to heat treatment, known as the Arrhenius effect (10, * This study was s...

DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the threedimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNAinteracting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.fidelity control | protein-DNA complex | replication fork | single particle analysis | structural bioinformatics

Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.