Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA.

The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

Drosophila model of Meier-Gorlin syndrome based on the mutation in a conserved C-Terminal domain of Orc6.

Meier-Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears, and skeletal abnormalities. Patients with MGS often carry mutations in the genes encoding the components of the pre-replicative complex such as Origin Recognition Complex (ORC) subunits Orc1, Orc4, Orc6, and helicase loaders Cdt1 and Cdc6. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. Mutation in conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. In order to study the effects of MGS mutation in an animal model system we introduced MGS mutation in Orc6 and established Drosophila model of MGS. Mutant flies die at third instar larval stage with abnormal chromosomes and DNA replication defects. The lethality can be rescued by elevated expression of mutant Orc6 protein. Rescued MGS flies are unable to fly and display multiple planar cell polarity defects. © 2015 Wiley Periodicals, Inc.

The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi.

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster. We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function.

Drosophila STING protein has a role in lipid metabolism.

Stimulator of interferon genes (STING) plays an important role in innate immunity by controlling type I interferon response against invaded pathogens. In this work, we describe a previously unknown role of STING in lipid metabolism in Drosophila. Flies with STING deletion are sensitive to starvation and oxidative stress, have reduced lipid storage, and downregulated expression of lipid metabolism genes. We found that Drosophila STING interacts with lipid synthesizing enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). ACC and FASN also interact with each other, indicating that all three proteins may be components of a large multi-enzyme complex. The deletion of Drosophila STING leads to disturbed ACC localization and decreased FASN enzyme activity. Together, our results demonstrate a previously undescribed role of STING in lipid metabolism in Drosophila.

Humanized Drosophila Model of the Meier-Gorlin Syndrome Reveals Conserved and Divergent Features of the Orc6 Protein.

Meier-Gorlin syndrome (MGS) is a rare, autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears, and skeletal abnormalities. Patients with MGS often carry mutations in genes encoding the subunits of the Origin Recognition Complex (ORC), components of the prereplicative complex and replication machinery. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. A mutation in the conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. Recently, a new mutation in Orc6 was also identified; however, it is localized in the N-terminal domain of the protein. To study the functions of Orc6, we used the human gene to rescue the orc6 deletion in Drosophila Using this "humanized" Orc6-based Drosophila model of MGS, we discovered that unlike the previous Y225S MGS mutation in Orc6, the K23E substitution in the N-terminal TFIIB-like domain of Orc6 disrupts the protein ability to bind DNA. Our studies revealed the importance of evolutionarily conserved and variable domains of Orc6 protein, and allowed the studies of human protein functions and the analysis of the critical amino acids in live animal heterologous system, as well as provided novel insights into the mechanisms underlying MGS pathology.

Phosphorylation of Pnut in the Early Stages of Drosophila Embryo Development Affects Association of the Septin Complex with the Membrane and Is Important for Viability.

Septin proteins are polymerizing GTPases that are found in most eukaryotic species. Septins are important for cytokinesis and participate in many processes involving spatial modifications of the cell cortex. In Drosophila, septin proteins Pnut, Sep1, and Sep2 form a hexameric septin complex. Here, we found that septin protein Pnut is phosphorylated during the first 2 hr of Drosophila embryo development. To study the effect of Pnut phosphorylation in a live organism, we created a new Drosophila pnut null mutant that allows for the analysis of Pnut mutations during embryogenesis. To understand the functional significance of Pnut phosphorylation, Drosophila strains carrying nonphosphorylatable and phospho-mimetic mutant pnut transgenes were established. The expression of the nonphosphorylatable Pnut protein resulted in semilethality and abnormal protein localization, whereas the expression of the phospho-mimetic mutant form of Pnut disrupted the assembly of a functional septin complex and septin filament formation in vitro Overall, our findings indicate that the controlled phosphorylation of Pnut plays an important role in regulating septin complex functions during organism development.

Functional insight into the role of Orc6 in septin complex filament formation in Drosophila.

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.