scholarly journals Topological in vitro loading of the budding yeast cohesin ring onto DNA

2018 ◽  
Vol 1 (5) ◽  
pp. e201800143 ◽  
Author(s):  
Masashi Minamino ◽  
Torahiko L Higashi ◽  
Céline Bouchoux ◽  
Frank Uhlmann
Keyword(s):  
Chromosome Segregation ◽  
Genome Organization ◽  
Atp Hydrolysis ◽  
Budding Yeast ◽  
Reaction Step ◽  
Subsequent Reaction ◽  
Transition State Analog ◽  
Sister Chromatids

The ring-shaped chromosomal cohesin complex holds sister chromatids together by topological embrace, a prerequisite for accurate chromosome segregation. Cohesin plays additional roles in genome organization, transcriptional regulation, and DNA repair. The cohesin ring includes an ABC family ATPase, but the molecular mechanism by which the ATPase contributes to cohesin function is not yet understood. In this study, we have purified budding yeast cohesin, as well as its Scc2–Scc4 cohesin loader complex, and biochemically reconstituted ATP-dependent topological cohesin loading onto DNA. Our results reproduce previous observations obtained using fission yeast cohesin, thereby establishing conserved aspects of cohesin behavior. Unexpectedly, we find that nonhydrolyzable ATP ground state mimetics ADP·BeF2, ADP·BeF3−, and ADP·AlFx, but not a hydrolysis transition state analog ADP·VO43−, support cohesin loading. The energy from nucleotide binding is sufficient to drive the DNA entry reaction into the cohesin ring. ATP hydrolysis, believed to be essential for in vivo cohesin loading, must serve a subsequent reaction step. These results provide molecular insights into cohesin function and open new experimental opportunities that the budding yeast model affords.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

Regulation of RNA helicase activity: principles and examples

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Pascal Donsbach ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Wide Spectrum ◽  
Mrna Translation ◽  
Rna Degradation ◽  
Rna Helicases ◽  
Interaction Partners ◽  
Rna Duplexes ◽  
Self Association

Abstract RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for the non-sequence-specific interactions with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

scholarly journals DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1357
Author(s):  
Rubén Torres ◽  
Carolina Gándara ◽  
Begoña Carrasco ◽  
Ignacio Baquedano ◽  
Silvia Ayora ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Holliday Junctions ◽  
Genome Integrity ◽  
Stable Complex ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Dna Structures ◽  
Checkpoint Protein

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c‑di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

scholarly journals Genes Involved in Sister Chromatid Separation and Segregation in the Budding Yeast Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 453-470
Author(s):  
Sue Biggins ◽  
Needhi Bhalla ◽  
Amy Chang ◽  
Dana L Smith ◽  
Andrew W Murray
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Mitotic Spindle ◽  
Budding Yeast ◽  
Temperature Sensitive ◽  
Chromosome Behavior ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sister Chromatids ◽  
Marked Chromosome ◽  
Sister Chromatid Separation

Abstract Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair α-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.

scholarly journals Pericentric chromatin loops function as a nonlinear spring in mitotic force balance

2013 ◽  
Vol 200 (6) ◽  
pp. 757-772 ◽  
Author(s):  
Andrew D. Stephens ◽  
Rachel A. Haggerty ◽  
Paula A. Vasquez ◽  
Leandra Vicci ◽  
Chloe E. Snider ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Force Balance ◽  
Nonlinear Spring ◽  
Sister Chromatids ◽  
Spindle Dynamics ◽  
Restoring Forces ◽  
Cross Links ◽  
Mean And Variance

The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes.

scholarly journals Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

2015 ◽  
Vol 290 (34) ◽  
pp. 21032-21041 ◽  
Author(s):  
Naman B. Shah ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Intrinsic Rate ◽  
Synthesis Rate ◽  
Final Segment ◽  
Rotary Catalysis

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

scholarly journals Overexpressed Pituitary Tumor-Transforming Gene Causes Aneuploidy in Live Human Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (11) ◽  
pp. 4991-4998 ◽  
Author(s):  
Run Yu ◽  
Wenge Lu ◽  
Jiandong Chen ◽  
Chris J. McCabe ◽  
Shlomo Melmed
Keyword(s):  
Cancer Cells ◽  
Chromosome Segregation ◽  
Pituitary Tumor ◽  
Fluorescent Protein ◽  
Human Cancer ◽  
Live Cells ◽  
Pituitary Tumor Transforming Gene ◽  
Transforming Gene

Abstract The mammalian securin, pituitary tumor-transforming gene (PTTG), is overexpressed in several tumors and transforms cells in vitro and in vivo. To test the hypothesis that PTTG overexpression causes aneuploidy, enhanced green fluorescent protein (EGFP)-tagged PTTG (PTTG-EGFP) was expressed in human H1299 cancer cells (with undetectable endogenous PTTG expression) and mitosis of individual live cells observed. Untransfected cells and cells expressing EGFP alone exhibited appropriate mitosis. PTTG-EGFP markedly prolonged prophase and metaphase, indicating that PTTG blocks progression of mitosis to anaphase. In cells that underwent apparently normal mitosis (35 of 65 cells), PTTG-EGFP was degraded about 1 min before anaphase onset. Cells that failed to degrade PTTG-EGFP exhibited asymmetrical cytokinesis without chromosome segregation (18 of 65 cells) or chromosome decondensation without cytokinesis (9 of 65 cells), resulting in appearance of a macronucleus. Fifty-one of 55 cells expressing a nondegradable mutant PTTG exhibited asymmetrical cytokinesis without chromosome segregation, and some (4 of 55) decondensed chromosomes, both resulting in macronuclear formation. During this abnormal cytokinesis, all chromosomes and spindles and both centrosomes moved to one daughter cell, suggesting potential chaos in the subsequent mitosis. In conclusion, failure of PTTG degradation or enhanced PTTG accumulation, as a consequence of overexpression, inhibits mitosis progression and chromosome segregation but does not directly affect cytokinesis, resulting in aneuploidy. These results demonstrate that PTTG induces aneuploidy in single, live, human cancer cells.

scholarly journals Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore.

1993 ◽  
Vol 121 (3) ◽  
pp. 513-519 ◽  
Author(s):  
W Jiang ◽  
J Lechner ◽  
J Carbon
Keyword(s):  
Molecular Motors ◽  
Cell Biology ◽  
Budding Yeast ◽  
Isolation And Characterization ◽  
Sequence Homologies ◽  
Binding Factor ◽  
Microtubule Motor

We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present. The CBF2 gene is essential for viability of yeast and is identical to NDC10, in which a conditional mutation leads to a defect in chromosome segregation (Goh, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.

scholarly journals A CDK-regulated chromatin segregase promoting chromosome replication

2020 ◽  
Author(s):  
Erika Chacin ◽  
Priyanka Bansal ◽  
Karl-Uwe Reusswig ◽  
Luis M. Diaz-Santin ◽  
Pedro Ortega ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Genome Instability ◽  
Chromosome Replication ◽  
S Phase ◽  
Chromatin Dynamics ◽  
Replicative Stress ◽  
Cdk Phosphorylation ◽  
Nucleosome Disassembly

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood.Here, we report the identification of a novel class of chromatin remodeling enzyme, entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+-ATPase that assembles into ~ 1 MDa hexameric complexes capable of segregating histones from DNA. Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that Yta7’s enzymatic activity is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication.Our results present a novel mechanism of how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

scholarly journals A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo

2021 ◽  
Vol 22 (22) ◽  
pp. 12502
Author(s):  
Shoji Kokubo ◽  
Shinobu Ohnuma ◽  
Megumi Murakami ◽  
Haruhisa Kikuchi ◽  
Shota Funayama ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cancer Cells ◽  
High Throughput Screening ◽  
Atp Hydrolysis ◽  
Pheophorbide A ◽  
Chemical Library ◽  
Hct 116 ◽  
Potential Inhibitors

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

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