Kouta Mayanagi

Crystal Structure of the RuvA-RuvB Complex: A Structural Basis for the Holliday Junction Migrating Motor Machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.

Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy

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.

Mechanism of replication machinery assembly as revealed by the DNA ligase–PCNA–DNA complex architecture

The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

The clamp-loading complex for processive DNA replication

DNA polymerase requires two processing factors, sliding clamps and clamp loaders, to direct rapid and accurate duplication of genomic DNA. In eukaryotes, proliferating cell nuclear antigen (PCNA), the ring-shaped sliding clamp, encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. Replication factor C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands in an ATP-dependent manner. Here we report the three-dimensional structure of an archaeal clamp-loading complex (RFC–PCNA–DNA) determined by single-particle EM. The three-dimensional structure of the complex, reconstituted in vitro using a nonhydrolyzable ATP analog, reveals two components, a closed ring and a horseshoe-shaped element, which correspond to PCNA and RFC, respectively. The atomic structure of PCNA fits well into the closed ring, suggesting that this ternary complex represents a state just after the PCNA ring has closed to encircle the DNA duplex.

Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

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 three-dimensional 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 PCNA-interacting 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.

Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases

Holliday junctions (HJs) are key intermediates in homologous recombination and are especially important for the production of crossover recombinants. Bacterial RecA family proteins promote the formation and branch migration of HJs in vitro by catalysing a reciprocal DNA-strand exchange reaction between two duplex DNA molecules, one of which contains a single-stranded DNA region that is essential for initial nucleoprotein filament formation. This activity has been reported only for prokaryotic RecA family recombinases, although eukaryotic homologues are also essential for HJ production in vivo. Here we show that fission yeast (Rhp51) and human (hRad51) RecA homologues promote duplex–duplex DNA-strand exchange in vitro. As with RecA, a HJ is formed between the two duplex DNA molecules, and reciprocal strand exchange proceeds through branch migration of the HJ. In contrast to RecA, however, strand exchange mediated by eukaryotic recombinases proceeds in the 3′→5′ direction relative to the single-stranded DNA region of the substrate DNA. The opposite polarity of Rhp51 makes it especially suitable for the repair of DNA double-strand breaks, whose repair is initiated at the processed ends of breaks that have protruding 3′ termini.

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.

Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma.

Structure and Functional Characterization of Vibrio parahaemolyticus Thermostable Direct Hemolysin

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 tetramer forms a central pore with dimensions of 23 Å in diameter and ∼50 Å in depth. π-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.

Structural determinant for switching between the polymerase and exonuclease modes in the PCNA-replicative DNA polymerase complex

Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with the cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction, with unprecedented configurations of the two molecules. Electron microscopic analyses indicated that this complex structure exists in solution. Our structural study revealed that an interaction occurs between a stretched loop of PCNA and the PfuPol Thumb domain, in addition to the authentic PCNA-polymerase recognition site (PIP box). Comparisons of the present structure with the previously reported structures of polymerases complexed with DNA, suggested that the second interaction plays a crucial role in switching between the polymerase and exonuclease modes, by inducing a PCNA-polymerase complex configuration that favors synthesis over editing. This putative mechanism for fidelity control of replicative DNA polymerases is supported by experiments, in which mutations at the second interaction site caused enhancements in the exonuclease activity in the presence of PCNA.