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The MIND multiprotein complex is a conserved, essential component of eukaryotic kinetochores and is a constituent of the tripartite KMN network that directly attaches the kinetochore to the mitotic spindle. The primary microtubule-binding complex in this network, NDC80, has been extensively characterized, but very little is known about the structure or function of the MIND complex. In this study, we present biochemical, hydrodynamic, electron microscopy, and small-angle x-ray scattering data that provide insight into the overall architecture and assembly of the MIND complex and the physical relationship of the complex with other components of the KMN network. We propose a model for the overall structure of the complex and provide data on the interactions with NDC80, Spc105p, and thus the mitotic spindle. doi/ 10.1083/jcb.201002059

The nomenclature of helicases and translocases which are highly diverse group of proteins that perform extraordinary variety of functions in cells are defined based on sequence, structure, and mechanism.

Author(s): Martin R. Singleton [1, 4]; Mark S. Dillingham [2, 4]; Martin Gaudier [1]; Stephen C. Kowalczykowski [3]; Dale B. Wigley (corresponding author) [1] Double-strand breaks in DNA are potentially [...]

The nucleic acid polymer translocating functionality of helicases is discussed. The two types of helicases, SuperFamily1 and SuperFamily2, appear to exhibit preference for single- or double-stranded substrates bound to the enzyme through the bases of phosphodiester backbone.

The processing of stalled DNA replication forks, due to DNA damage, is mediated by RecG protein. Data indicate that RecG reverses the fork past the damage and forms a four-way junction, which pemits template switching and lesion bypass.

The crystal structure of T7 gene 4 ring helicase has been found to indicate a mechanism for sequential nucleotide hydrolysis. The crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7 has been determined.

Abstract: Eukaryotic chromosomes contain a specialised region known as the centromere, which forms the platform for kinetochore assembly and microtubule attachment. The centromere is distinguished by the presence of nucleosomes containing the histone H3 variant, CENP‐A. In budding yeast, centromere establishment begins with the recognition of a specific DNA sequence by the CBF3 complex. This in turn facilitates CENP‐ACse4 nucleosome deposition and kinetochore assembly. Here, we describe a 3.6 Å single‐particle cryo‐EM reconstruction of the core CBF3 complex, incorporating the sequence‐specific DNA‐binding protein Cep3 together with regulatory subunits Ctf13 and Skp1. This provides the first structural data on Ctf13, defining it as an F‐box protein of the leucine‐rich‐repeat family, and demonstrates how a novel F‐box‐mediated interaction between Ctf13 and Skp1 is responsible for initial assembly of the CBF3 complex. [ABSTRACT FROM AUTHOR]

Replication factor C complexes load and unload processivity clamps from DNA and are involved in multiple DNA replication and repair pathways. The RFCCtf18 variant complex is required for activation of the intra-S-phase checkpoint at stalled replication forks and aids the establishment of sister chromatid cohesion. Unlike other RFC complexes, RFCCtf18 contains two non-Rfc subunits, Dcc1 and Ctf8. Here, we present the crystal structure of the Dcc1-Ctf8 heterodimer bound to the C-terminus of Ctf18. We find that the C-terminus of Dcc1 contains three-winged helix domains, which bind to both ss DNA and ds DNA. We further show that these domains are required for full recruitment of the complex to chromatin, and correct activation of the replication checkpoint. These findings provide the first structural data on a eukaryotic seven-subunit clamp loader and define a new biochemical activity for Dcc1. [ABSTRACT FROM AUTHOR]

Dynamic microtubule plus-ends interact with various intracellular target regions such as the cell cortex and the kinetochore. Two conserved families of microtubule plus-end-tracking proteins, the XMAP215, ch-TOG or CKAP5 family and the end-binding 1 (EB1, also known as MAPRE1) family, play pivotal roles in regulating microtubule dynamics. Here, we study the functional interplay between fission yeast Dis1, a member of the XMAP215/TOG family, and Mal3, an EB1 protein. Using an in vitro microscopy assay, we find that purified Dis1 autonomously tracks growing microtubule ends and is a bona fide microtubule polymerase. Mal3 recruits additional Dis1 to microtubule ends, explaining the synergistic enhancement of microtubule dynamicity by these proteins. A non-canonical binding motif in Dis1 mediates the interaction with Mal3. X-ray crystallography shows that this new motif interacts in an unconventional configuration with the conserved hydrophobic cavity formed within the Mal3 C-terminal region that typically interacts with the canonical SXIP motif. Selectively perturbing the Mal3-Dis1 interaction in living cells demonstrates that it is important for accurate chromosome segregation. Whereas, in some metazoans, the interaction between EB1 and the XMAP215/TOG family members requires an additional binding partner, fission yeast relies on a direct interaction, indicating evolutionary plasticity of this critical interaction module. [ABSTRACT FROM AUTHOR]

The Ndc80 complex establishes end-on attachment of kinetochores to microtubules, which is essential for chromosome segregation. The Ndc80 subunit is characterized by an N-terminal region that binds directly to microtubules, and a long coiled-coil region that interacts with Nuf2. A loop region in Ndc80 that generates a kink in the structure disrupts the long coiled-coil region but the exact function of this loop, has until now, not been clear. Here we show that this loop region is essential for end-on attachment of kinetochores to microtubules in human cells. Cells expressing loop mutants of Ndc80 are unable to align the chromosomes, and stable kinetochore fibers are absent. Through quantitative mass spectrometry and immunofluorescence we found that the binding of the spindle and kinetochore associated (Ska) complex depends on the loop region, explaining why end-on attachment is defective. This underscores the importance of the Ndc80 loop region in coordinating chromosome segregation through the recruitment of specific proteins to the kinetochore. [ABSTRACT FROM AUTHOR]