scholarly journals Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome

2011 ◽  
Vol 195 (4) ◽  
pp. 573-582 ◽  
Author(s):  
Josh Lawrimore ◽  
Kerry S. Bloom ◽  
E.D. Salmon
Keyword(s):  
Fusion Proteins ◽  
Copy Number ◽  
Budding Yeast ◽  
Specific Binding ◽  
Histone H3 ◽  
Attachment Site ◽  
Microtubule Attachment ◽  
Copy Numbers ◽  
Gfp Fluorescence

Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ∼5 Cse4s, ∼3 inner kinetochore CBF3 complexes, and ∼20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5–3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.

scholarly journals Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

1997 ◽  
Vol 328 (2) ◽  
pp. 669-675 ◽  
Author(s):  
L. Tamara DOERING ◽  
Randy SCHEKMAN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Proteins ◽  
Attachment Site ◽  
Gpi Anchor ◽  
Mating Pheromone ◽  
Peptide Substrates ◽  
In Vitro System ◽  
Glycosyl Phosphatidylinositol

The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.

scholarly journals Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres

2008 ◽  
Vol 181 (4) ◽  
pp. 587-594 ◽  
Author(s):  
Ajit P. Joglekar ◽  
David Bouck ◽  
Ken Finley ◽  
Xingkun Liu ◽  
Yakun Wan ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Budding Yeast ◽  
Attachment Site ◽  
Molecular Architecture ◽  
Kinetochore Protein ◽  
Kinetochore Assembly ◽  
Microtubule Attachment ◽  
Unique Site ◽  
Structural Subunit ◽  
Quantitative Fluorescence

Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

2004 ◽  
Vol 186 (18) ◽  
pp. 6254-6264 ◽  
Author(s):  
Peggy Freede ◽  
Sabine Brantl
Keyword(s):  
Binding Affinity ◽  
Copy Number ◽  
Dissociation Constants ◽  
Mobility Shift ◽  
Copy Numbers ◽  
Maximal Binding ◽  
Equilibrium Dissociation Constants ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5′-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 μM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3′ positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

scholarly journals CENP-A exceeds microtubule attachment sites in centromere clusters of both budding and fission yeast

2011 ◽  
Vol 195 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Valerie C. Coffman ◽  
Pengcheng Wu ◽  
Mark R. Parthun ◽  
Jian-Qiu Wu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Histone H3 ◽  
Kinetochore Assembly ◽  
Microtubule Attachment ◽  
Attachment Sites ◽  
Histone H3 Variant ◽  
Architectural Models

The stoichiometries of kinetochores and their constituent proteins in yeast and vertebrate cells were determined using the histone H3 variant CENP-A, known as Cse4 in budding yeast, as a counting standard. One Cse4-containing nucleosome exists in the centromere (CEN) of each chromosome, so it has been assumed that each anaphase CEN/kinetochore cluster contains 32 Cse4 molecules. We report that anaphase CEN clusters instead contained approximately fourfold more Cse4 in Saccharomyces cerevisiae and ∼40-fold more CENP-A (Cnp1) in Schizosaccharomyces pombe than predicted. These results suggest that the number of CENP-A molecules exceeds the number of kinetochore-microtubule (MT) attachment sites on each chromosome and that CENP-A is not the sole determinant of kinetochore assembly sites in either yeast. In addition, we show that fission yeast has enough Dam1–DASH complex for ring formation around attached MTs. The results of this study suggest the need for significant revision of existing CEN/kinetochore architectural models.

scholarly journals The structure of the Ctf19c/CCAN from budding yeast

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Stephen M Hinshaw ◽  
Stephen C Harrison
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Budding Yeast ◽  
Histone H3 ◽  
Kinetochore Assembly ◽  
Microtubule Attachment ◽  
Cryo Electron Microscopy ◽  
Histone H3 Variant

Eukaryotic kinetochores connect spindlemicrotubules to chromosomal centromeres. A group of proteins called the Ctf19 complex (Ctf19c) in yeast and the constitutive centromere associated network (CCAN) in other organisms creates the foundation of a kinetochore. The Ctf19c/CCAN influences the timing of kinetochore assembly, sets its location by associating with a specialized nucleosome containing the histone H3 variant Cse4/CENP-A, and determines the organization of the microtubule attachment apparatus. We present here the structure of a reconstituted 13-subunit Ctf19c determined by cryo-electron microscopy at ~4 Å resolution. The structure accounts for known and inferred contacts with the Cse4 nucleosome and for an observed assembly hierarchy. We describe its implications for establishment of kinetochores and for their regulation by kinases throughout the cell cycle.

scholarly journals Lamin B constitutes an intermediate filament attachment site at the nuclear envelope.

1987 ◽  
Vol 105 (1) ◽  
pp. 117-125 ◽  
Author(s):  
S D Georgatos ◽  
G Blobel
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Attachment Site ◽  
Nuclear Lamina ◽  
Membrane Skeleton ◽  
Eukaryotic Cells ◽  
Lamin A ◽  
Lamin B ◽  
Nuclear Lamins

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero-oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti-lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.

scholarly journals Bifunctional Role of theTreponema pallidumExtracellular Matrix Binding Adhesin Tp0751

2010 ◽  
Vol 79 (3) ◽  
pp. 1386-1398 ◽  
Author(s):  
Simon Houston ◽  
Rebecca Hof ◽  
Teresa Francescutti ◽  
Aaron Hawkes ◽  
Martin J. Boulanger ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Specific Binding ◽  
Circulatory System ◽  
Attachment Site ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Active Site Residues ◽  
Human Fibrinogen

ABSTRACTTreponema pallidum, the causative agent of syphilis, is a highly invasive pathogenic spirochete capable of attaching to host cells, invading the tissue barrier, and undergoing rapid widespread dissemination via the circulatory system. TheT. pallidumadhesin Tp0751 was previously shown to bind laminin, the most abundant component of the basement membrane, suggesting a role for this adhesin in host tissue colonization and bacterial dissemination. We hypothesized that similar to that of other invasive pathogens, the interaction ofT. pallidumwith host coagulation proteins, such as fibrinogen, may also be crucial for dissemination via the circulatory system. To test this prediction, we used enzyme-linked immunosorbent assay (ELISA) methodology to demonstrate specific binding of soluble recombinant Tp0751 to human fibrinogen. Click-chemistry-based palmitoylation profiling of heterologously expressed Tp0751 confirmed the presence of a lipid attachment site within this adhesin. Analysis of the Tp0751 primary sequence revealed the presence of a C-terminal putative HEXXH metalloprotease motif, andin vitrodegradation assays confirmed that recombinant Tp0751 purified from both insect andEscherichia coliexpression systems degrades human fibrinogen and laminin. The proteolytic activity of Tp0751 was abolished by the presence of the metalloprotease inhibitor 1,10-phenanthroline. Further, inductively coupled plasma-mass spectrometry showed that Tp0751 binds zinc and calcium. Collectively, these results indicate that Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, site-directed mutagenesis of the HEXXH motif to HQXXH did not abolish the proteolytic activity of Tp0751, indicating that further mutagenesis studies are required to elucidate the critical active site residues associated with this protein. This study represents the first published description of aT. pallidumprotease capable of degrading host components and thus provides novel insight into the mechanism ofT. pallidumdissemination.

scholarly journals The Role of Human Satellite III (1q12) Copy Number Variation in the Adaptive Response during Aging, Stress, and Pathology: A Pendulum Model

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1524
Author(s):  
Lev N. Porokhovnik ◽  
Natalia N. Veiko ◽  
Elizaveta S. Ershova ◽  
Svetlana V. Kostyuk
Keyword(s):  
Stress Response ◽  
Copy Number Variation ◽  
Copy Number ◽  
Tandem Repeats ◽  
Genotoxic Stress ◽  
Close Link ◽  
Copy Numbers ◽  
Number Variation

The pericentric satellite III (SatIII or Sat3) and II tandem repeats recently appeared to be transcribed under stress conditions, and the transcripts were shown to play an essential role in the universal stress response. In this paper, we review the role of human-specific SatIII copy number variation (CNV) in normal stress response, aging and pathology, with a focus on 1q12 loci. We postulate a close link between transcription of SatII/III repeats and their CNV. The accrued body of data suggests a hypothetical universal mechanism, which provides for SatIII copy gain during the stress response, alongside with another, more hypothetical reverse mechanism that might reduce the mean SatIII copy number, likely via the selection of cells with excessively large 1q12 loci. Both mechanisms, working alternatively like swings of the pendulum, may ensure the balance of SatIII copy numbers and optimum stress resistance. This model is verified on the most recent data on SatIII CNV in pathology and therapy, aging, senescence and response to genotoxic stress in vitro.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2019 ◽  
Author(s):  
Erin Cutts ◽  
J. Barry Egan ◽  
Ian Dodd ◽  
Keith Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

AbstractThe Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA, and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl’s repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

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