scholarly journals CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

2019 ◽  
Author(s):  
N Cortes-Silva ◽  
J Ulmer ◽  
T Kiuchi ◽  
E Hsieh ◽  
G Cornilleau ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Gene Editing ◽  
Mitotic Progression ◽  
Critical Function ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Essential Function ◽  
Necessary And Sufficient ◽  
Assembly Pathways

AbstractAccurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

scholarly journals The ATAD2/ANCCA homolog Yta7 cooperates with Scm3HJURP to deposit Cse4CENP-A at the centromere in yeast

2020 ◽  
Vol 117 (10) ◽  
pp. 5386-5393 ◽  
Author(s):  
Sara Shahnejat-Bushehri ◽  
Ann E. Ehrenhofer-Murray
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Histone H3 ◽  
Nucleosome Assembly ◽  
Direct Role ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Assembly Factor

The AAA+ ATPase and bromodomain factor ATAD2/ANCCA is overexpressed in many types of cancer, but how it contributes to tumorigenesis is not understood. Here, we report that the Saccharomyces cerevisiae homolog Yta7ATAD2 is a deposition factor for the centromeric histone H3 variant Cse4CENP-A at the centromere in yeast. Yta7ATAD2 regulates the levels of centromeric Cse4CENP-A in that yta7∆ causes reduced Cse4CENP-A deposition, whereas YTA7 overexpression causes increased Cse4CENP-A deposition. Yta7ATAD2 coimmunoprecipitates with Cse4CENP-A and is associated with the centromere, arguing for a direct role of Yta7ATAD2 in Cse4CENP-A deposition. Furthermore, increasing centromeric Cse4CENP-A levels by YTA7 overexpression requires the activity of Scm3HJURP, the centromeric nucleosome assembly factor. Importantly, Yta7ATAD2 interacts in vivo with Scm3HJURP, indicating that Yta7ATAD2 is a cochaperone for Scm3HJURP. The absence of Yta7 causes defects in growth and chromosome segregation with mutations in components of the inner kinetochore (CTF19/CCAN, Mif2CENP-C, Cbf1). Since Yta7ATAD2 is an AAA+ ATPase and potential hexameric unfoldase, our results suggest that it may unfold the Cse4CENP-A histone and hand it over to Scm3HJURP for subsequent deposition in the centromeric nucleosome. Furthermore, our findings suggest that ATAD2 overexpression may enhance malignant transformation in humans by misregulating centromeric CENP-A levels, thus leading to defects in kinetochore assembly and chromosome segregation.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

scholarly journals Molecular requirements for the formation of a kinetochore–microtubule interface by Dam1 and Ndc80 complexes

2012 ◽  
Vol 200 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Fabienne Lampert ◽  
Christine Mieck ◽  
Gregory M. Alushin ◽  
Eva Nogales ◽  
Stefan Westermann
Keyword(s):  
Chromosome Segregation ◽  
Protein Complexes ◽  
Structural Elements ◽  
Large Protein ◽  
Kinetochore Assembly ◽  
Sister Chromatids ◽  
Dam1 Complex ◽  
Shed Light ◽  
Kinetochore Function

Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore–microtubule interface is formed by the plus end–associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80–Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80–Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.

scholarly journals Decoding the centromeric nucleosome through CENP-N

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Satyakrishna Pentakota ◽  
Keda Zhou ◽  
Charlotte Smith ◽  
Stefano Maffini ◽  
Arsen Petrovic ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Double Helix ◽  
Structural Basis ◽  
Binding Motif ◽  
Kinetochore Assembly ◽  
Nucleosomal Dna ◽  
Chromosome Domains ◽  
Histone H3 Variant ◽  
Spindle Microtubules ◽  
Dna Double Helix

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.

scholarly journals A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Pooja Sharma ◽  
Robert Mahen ◽  
Maxim Rossmann ◽  
Jamie E. Stokes ◽  
Bryn Hardwick ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Substrate Recognition ◽  
Genome Integrity ◽  
Mitotic Progression ◽  
New Approach ◽  
Molecule Inhibitor ◽  
Evolutionarily Conserved ◽  
Essential Function ◽  
Carboxyl Terminal

Abstract The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.

Topological similarity between the 2μm plasmid partitioning locus and the budding yeast centromere: evidence for a common evolutionary origin?

2013 ◽  
Vol 41 (2) ◽  
pp. 501-507 ◽  
Author(s):  
Makkuni Jayaram ◽  
Keng-Ming Chang ◽  
Chien-Hui Ma ◽  
Chu-Chun Huang ◽  
Yen-Ting Liu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Host Factors ◽  
Dependent Manner ◽  
Chromosomal Dna ◽  
Topological Similarity ◽  
Plasmid Partitioning ◽  
Histone H3 Variant ◽  
Functional States ◽  
Positive Supercoiling

The partitioning locus STB of the selfish plasmid, the 2μm circle, of Saccharomyces cerevisiae is essential for the propagation of this multi-copy extra-chromosomal DNA element with nearly chromosome-like stability. The functional competence of STB requires the plasmid-coded partitioning proteins Rep1 and Rep2 as well as host-coded proteins. Host factors that associate with STB in a Rep1- and Rep2-dependent manner also interact with centromeres, and play important roles in chromosome segregation. They include the cohesin complex and the centromere-specific histone H3 variant Cse4. The genetically defined point centromere of S. cerevisiae differs starkly from the much more widespread epigenetically specified regional centromeres of eukaryotes. The particularly small size of the S. cerevisiae centromere and the association of chromosome segregation factors with STB raise the possibility of an evolutionary link between these two partitioning loci. The unusual positive supercoiling harboured by the S. cerevisiae centromere and STB in vivo in their functional states, unveiled by recent experiments, bolsters the notion of their potential descent from an ancestral plasmid partitioning locus.

scholarly journals Cell cycle–dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast

2019 ◽  
Vol 30 (8) ◽  
pp. 1020-1036 ◽  
Author(s):  
Prashant K. Mishra ◽  
Gudjon Olafsson ◽  
Lars Boeckmann ◽  
Timothy J. Westlake ◽  
Ziad M. Jowhar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Growth Defects ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant ◽  
Cycle Dependent

Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle–dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle–regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle–regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.

scholarly journals The negatively charged carboxy-terminal tail of β-tubulin promotes proper chromosome segregation

2016 ◽  
Vol 27 (11) ◽  
pp. 1786-1796 ◽  
Author(s):  
Colby P. Fees ◽  
Jayne Aiken ◽  
Eileen T. O’Toole ◽  
Thomas H. Giddings ◽  
Jeffrey K. Moore
Keyword(s):  
Chromosome Segregation ◽  
Electron Tomography ◽  
Chromosome Loss ◽  
Molecular Features ◽  
Charged Amino Acids ◽  
Spindle Poles ◽  
Underlying Mechanisms ◽  
Carboxy Terminal ◽  
Necessary And Sufficient

Despite the broadly conserved role of microtubules in chromosome segregation, we have a limited understanding of how molecular features of tubulin proteins contribute to the underlying mechanisms. Here we investigate the negatively charged carboxy-terminal tail domains (CTTs) of α- and β-tubulins, using a series of mutants that alter or ablate CTTs in budding yeast. We find that ablating β-CTT causes elevated rates of chromosome loss and cell cycle delay. Complementary live-cell imaging and electron tomography show that β-CTT is necessary to properly position kinetochores and organize microtubules within the assembling spindle. We identify a minimal region of negatively charged amino acids that is necessary and sufficient for proper chromosome segregation and provide evidence that this function may be conserved across species. Our results provide the first in vivo evidence of a specific role for tubulin CTTs in chromosome segregation. We propose that β-CTT promotes the ordered segregation of chromosomes by stabilizing the spindle and contributing to forces that move chromosomes toward the spindle poles.

scholarly journals Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

2013 ◽  
Vol 24 (12) ◽  
pp. 2034-2044 ◽  
Author(s):  
Lars Boeckmann ◽  
Yoshimitsu Takahashi ◽  
Wei-Chun Au ◽  
Prashant K. Mishra ◽  
John S. Choy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Histone H3 ◽  
In Vitro Assays ◽  
Histone H3 Variant

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

scholarly journals A Rad51-Independent Pathway Promotes Single-Strand Template Repair in Gene Editing

2020 ◽  
Author(s):  
Danielle N. Gallagher ◽  
Nhung Pham ◽  
Annie M. Tsai ◽  
Abigail N. Janto ◽  
Jihyun Choi ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Gene Editing ◽  
Fold Increase ◽  
Single Strand ◽  
Critical Function ◽  
Strand Breaks ◽  
Independent Events ◽  
Single Strand Annealing ◽  
Strand Annealing

AbstractThe Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to facilitate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae in two different assays. Gene editing events were carried out either by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs) or by using a bacterial retron system that produces ssDNA templates in vivo in combination with DSBs created by Cas9. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but not Rad51, Rad54 or Rad55. Srs2 acts to prevent overloading of Rad51 on the ssDNA filament, whereas Rad59 appears to alleviate the inhibition of Rad51 on Rad52’s strand annealing activity; thus, deletion of RAD51 suppresses both the srs2Δ and rad59Δ phenotypes. This same suppression by rad51Δ of rad59Δ is found in another DSB repair pathway, single strand annealing (SSA). In contrast, gene targeting using an 80-bp dsDNA template of the same sequence is Rad51-dependent. We also examined SSTR events in which the ssODN carried several mismatches. In the absence of the mismatch repair protein, Msh2, we found that the fate of mismatches carried on the ssDNA template are very different at the 3’ end, which can anneal directly to the resected DSB end, compared to the 5’ end. We also find that DNA polymerase Polδ’s 3’ to 5’ proofreading activity frequently excises a mismatch close to the 3’ end of the template, similar to its removal of heterologies close to the 3’ invading end of the DSB. We further report that SSTR is accompanied by a 600-fold increase in mutations in a region adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

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