suGF1 binds in the major groove of its oligo(dG).oligo(dC) recognition sequence and is excluded by a positioned nucleosome core

We have elsewhere reported the purification of a poly(dG).poly(dC)-binding nuclear protein (suGF1) from sea urchin embryos (J. Hapgood and D. Patterton, Mol. Cell. Biol. 14:this issue, 1994). We proposed that suGF1 may be a member of a family of G-string factors involved in developmental gene regulation, possibly via alterations in chromatin structure. In this article, we characterize the binding of purified suGF1 to 11 contiguous Gs in the H1-H4 intergenic region of a sea urchin early histone gene battery in vitro. It is shown that suGF1-DNA binding is dependent on ionic strength and requires divalent cations. Purified suGF1 forms discrete protein-DNA multimers, consistent with suGF1-suGF1 interactions. In a model for the suGF1-DNA complex derived from our footprinting and methylation interference data, suGF1 contacts the Gs in the major groove as well as one of the bordering phosphate backbones. The data are consistent with the direction of curvature of the DNA in the suGF1-DNA complex being the same as that preferred by the free DNA and exhibited by the DNA when bent around a positioned nucleosome core in vitro. However, on the basis of steric considerations, the binding of suGF1 and that of the histone octamer are predicted to be mutually exclusive. We show that suGF1 is indeed unable to bind to the G string when occupied by a histone octamer located in the major in vitro positioning frame in the H1-H4 intergenic region.

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The prenucleosome, a stable conformational isomer of the nucleosome

Genes & Development ◽

10.1101/gad.272633.115 ◽

2015 ◽

Vol 29 (24) ◽

pp. 2563-2575 ◽

Cited By ~ 2

Author(s):

Jia Fei ◽

Sharon E. Torigoe ◽

Christopher R. Brown ◽

Mai T. Khuong ◽

George A. Kassavetis ◽

...

Keyword(s):

Motor Protein ◽

Upstream Region ◽

Entry And Exit ◽

Core Particle ◽

Nucleosome Core Particle ◽

Nucleosome Core ◽

Histone Octamer ◽

Conformational Isomer

Chromatin comprises nucleosomes as well as nonnucleosomal histone–DNA particles. Prenucleosomes are rapidly formed histone–DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone–DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone–DNA species in the cell.

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RAD51AP1 mediates RAD51 activity through nucleosome interaction

10.1101/2020.12.17.421636 ◽

2020 ◽

Author(s):

Elena Pires ◽

Neelam Sharma ◽

Platon Selemenakis ◽

Bo Wu ◽

Yuxin Huang ◽

...

Keyword(s):

Genome Instability ◽

Loop Formation ◽

Synaptic Complex ◽

Nucleosome Core ◽

Histone Octamer ◽

Nucleosome Core Particles ◽

Dna Capture ◽

Dna Template ◽

Template Dna

AbstractRAD51 Associated Protein 1 (RAD51AP1) is a key protein in the homologous recombination DNA repair pathway (HR). Loss of RAD51AP1 leads to defective HR, genome instability and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of the donor DNA, synaptic complex assembly and displacement-loop formation when tested with synthetic, nucleosome-free DNA substratesin vitro. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. How RAD51AP1 functions as an HR activator in the context of chromatin has remained unclear.In this study, we show that RAD51AP1 binds to Nucleosome Core Particles (NCPs). We identified a C-terminal region in RAD51AP1 and its previously mapped DNA binding domain as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. We show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1directlyassists the RAD51-mediated search of donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

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A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo.

The Journal of Cell Biology ◽

10.1083/jcb.109.3.1289 ◽

1989 ◽

Vol 109 (3) ◽

pp. 1289-1299 ◽

Cited By ~ 27

Author(s):

M C Farach-Carson ◽

D D Carson ◽

J L Collier ◽

W J Lennarz ◽

H R Park ◽

...

Keyword(s):

Sea Urchin ◽

Calcium Binding ◽

Divalent Cations ◽

Surface Protein ◽

Oligosaccharide Chain ◽

Oligosaccharide Moiety ◽

Mesenchyme Cells ◽

Skeleton Formation ◽

Primary Mesenchyme Cells

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.

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Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro

Development ◽

10.1242/dev.101.2.297 ◽

1987 ◽

Vol 101 (2) ◽

pp. 297-312 ◽

Cited By ~ 5

Author(s):

G.L. Decker ◽

J.B. Morrill ◽

W.J. Lennarz

Keyword(s):

Electron Microscopy ◽

Sea Urchin ◽

Secretory Pathway ◽

Divalent Cations ◽

Ruthenium Red ◽

Coated Pits ◽

Residual Matrix ◽

Mesenchyme Cells ◽

Primary Mesenchyme Cells

An in vitro culture system for primary mesenchyme cells of the sea urchin embryo has been used to study the cellular characteristics of skeletal spicule formation. As judged initially by light microscopy, these cells attached to plastic substrata, migrated and fused to form syncytia in which mineral deposits accumulated in the cell bodies and in specialized filopodial templates. Subsequent examination by scanning electron microscopy revealed that the cell bodies and the filopodia and lamellipodia formed spatial associations similar to those seen in the embryo and indicated that the spicule was surrounded by a membrane-limited sheath derived by fusion of the filopodia. The spicules were dissolved from living or fixed cells by a chelator of divalent cations or by lowering the pH of the medium. However, granular deposits found in the cell bodies appeared relatively refractory to such treatments, indicating that they were inaccessible to agents that dissolved the spicules. Use of rapid freezing and an anhydrous fixative to preserve the syncytia for transmission electron microscopy and X-ray microprobe analysis, indicated that electron-dense deposits in the cell bodies contain elements (Ca, Mg and S) common to the spicule. Examination of the spicule cavity after dissolution of the spicule mineral revealed openings in the filopodia-derived sheath, coated pits within the limiting membrane and a residual matrix that stained with ruthenium red. Concanavalin A—gold applied exogenously entered the spicule cavity and bound to matrix glycoproteins. Based on these observations, we conclude that components of the spicule initially are sequestered intracellularly and that spicule elongation occurs in an extracellular cavity. Ca2+ and associated glycoconjugates may be routed in this cavity via a secretory pathway.

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The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Nature Communications ◽

10.1038/s41467-021-24418-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Brianna J. Klein ◽

Anagha Deshpande ◽

Khan L. Cox ◽

Fan Xuan ◽

Mohamad Zandian ◽

...

Keyword(s):

Critical Role ◽

Cellular Transformation ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Nucleosome Core ◽

Stem And Progenitor Cells ◽

Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

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Structural characterization and immunostimulatory activity in vitro of a glycogen from sea urchin–Strongylocentyotus internedius

Carbohydrate Polymers ◽

10.1016/j.carbpol.2021.117701 ◽

2021 ◽

Vol 258 ◽

pp. 117701

Author(s):

Qing-Chi Wang ◽

Maosheng Wei ◽

Yang Yue ◽

Ning Wu ◽

Jing Wang ◽

...

Keyword(s):

Sea Urchin ◽

Structural Characterization ◽

Immunostimulatory Activity

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In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB

International Journal of Molecular Sciences ◽

10.3390/ijms22147455 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7455

Author(s):

Bini Chhetri Soren ◽

Jagadish Babu Dasari ◽

Alessio Ottaviani ◽

Beatrice Messina ◽

Giada Andreotti ◽

...

Keyword(s):

Enzyme Reaction ◽

Dna Topoisomerase ◽

Human Dna ◽

Topoisomerase Ib ◽

Topological State ◽

Cleavage Step ◽

Dynamics Simulations ◽

In Silico Characterization ◽

Dna Complex

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.

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Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

BioMed Research International ◽

10.1155/2015/965804 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 11

Author(s):

Pia Montanucci ◽

Silvia Terenzi ◽

Claudio Santi ◽

Ilaria Pennoni ◽

Vittorio Bini ◽

...

Keyword(s):

Divalent Cations ◽

Chemical Properties ◽

Live Cells ◽

High Performing ◽

Physical Chemical ◽

Degenerative Disorders ◽

Selection Of ◽

Better Than

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

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