E. coli MukB protein involved in chromosome partition forms a homodimer with a rod-and-hinge structure having DNA binding ATP/GTP binding activities

Department of Molecular Cell Biology, Kumamoto University School of Medicine, Japan.
The EMBO Journal (Impact Factor: 10.75). 01/1993; 11(13):5101-9.
Source: PubMed

ABSTRACT mukB mutants of Escherichia coli are defective in the correct partitioning of replicated chromosomes. This results in the appearance of normal-sized anucleate (chromosome-less) cells during cell proliferation. Based on the nucleotide sequence of the mukB gene, the MukB protein of 177 kDa was predicted to be a filamentous protein with globular domains at the ends, and also having DNA binding and nucleotide binding abilities. Here we present evidence that the purified MukB protein possesses these characteristics. MukB forms a homodimer with a rod-and-hinge structure having a pair of large, C-terminal globular domains at one end and a pair of small, N-terminal globular domains at the opposite end; it tends to bend at a middle hinge site of the rod section. Chromatography in a DNA-cellulose column and the gel retardation assay revealed that MukB possesses DNA binding activity. Photoaffinity cross-linking experiments showed that MukB binds to ATP and GTP in the presence of Zn2+. Throughout the purification steps, acyl carrier protein was co-purified with MukB.

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Available from: Hironori Niki, Aug 05, 2015
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    • "Slimfield microscopy can also be used in dual-colour fluorescence microscopy and has been used to study the bacterial SMC proteins in E. coli.[11] SMC proteins have conserved architecture and function across all domains of life with bacteria using a distant relative called MukB with accessory MukE and MukF proteins playing a role in chromosome segregation and organization.[15] [16] Structural and biochemical studies have shown two stoichiometries for the MukBEF complex of 2:4:2 and 2:2:1 (MukB:E:F) dependent on whether ATP is bound or unbound.[17] "
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    ABSTRACT: DNA-interacting proteins have roles multiple processes, many operating as molecular machines which undergo dynamic metastable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner fast to sample the molecular transitions across the free energy landscape. Progress has been made over the past decade in utilising cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging, but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection single-to-noise of molecular signatures and transition events. Here, we discuss a range of techniques we have developed to monitor DNA-protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our lab made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new superresolution fluorescence microscope.
    Biochemical Society Transactions 04/2015; 43(2):139. DOI:10.1042/BST20140253 · 3.24 Impact Factor
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    • "Strikingly, the STAS domain expressed in E. coli copurified in a 1:1 complex with acyl carrier protein (ACP) and its high-resolution crystal structure revealed a specific interaction between ACP and the STAS domain of YchM. In E. coli, ACP is an abundant 77 residue protein (0.25% of total soluble protein, $5 3 10 4 molecules/cell) that runs anomalously slowly upon SDS-PAGE gels (Byers and Gong, 2007a; Niki et al., 1992). ACP contains a 4 0 -phosphopantetheine group (4 0 -PPa) covalently attached to Ser36 that acts as an activated thiol ester carrier of acyl intermediates during fatty acid biosynthesis (FAB) and other acylation reactions (Byers and Gong, 2007b) such as the biosynthesis of lipid A (Anderson and Raetz, 1987) and phospholipids (Rock and Jackowski, 1982). "
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    Structure 11/2010; 18(11):1450-62. DOI:10.1016/j.str.2010.08.015 · 6.79 Impact Factor
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    • "We examined here DNA bridging by MukBEF. MukBEF plays a key role in organizing the chromosome of Escherichia coli (Niki et al, 1992; Yamanaka et al, 1996; Sawitzke and Austin, 2000; Danilova et al, 2007; She et al, 2007) and is a bacterial prototype of eukaryotic condensins and cohesins. "
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    ABSTRACT: Structural maintenance of chromosome (SMC) proteins comprise the core of several specialized complexes that stabilize the global architecture of the chromosomes by dynamically linking distant DNA fragments. This reaction however remains poorly understood giving rise to numerous proposed mechanisms of the proteins. Using two novel assays, we investigated real-time formation of DNA bridges by bacterial condensin MukBEF. We report that MukBEF can efficiently bridge two DNAs and that this reaction involves multiple steps. The reaction begins with the formation of a stable MukB-DNA complex, which can further capture another protein-free DNA fragment. The initial tether is unstable but is quickly strengthened by additional MukBs. DNA bridging is modulated but is not strictly dependent on ATP and MukEF. The reaction revealed high preference for right-handed DNA crossings indicating that bridging involves physical association of MukB with both DNAs. Our data establish a comprehensive view of DNA bridging by MukBEF, which could explain how SMCs establish both intra- and interchromosomal links inside the cell and indicate that DNA binding and bridging could be separately regulated.
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