scholarly journals Mechanistic Insights into Replication Termination as Revealed by Investigations of the Reb1-Ter3 Complex of Schizosaccharomyces pombe

2008 ◽  
Vol 28 (22) ◽  
pp. 6844-6857 ◽  
Author(s):  
Subhrajit Biswas ◽  
Deepak Bastia
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Replication Fork ◽  
Intrinsic Property ◽  
Dimerization Domain ◽  
Mammalian Counterpart ◽  
Chromosome Iii ◽  
Necessary And Sufficient ◽  
Replication Fork Arrest

ABSTRACT Relatively little is known about the interaction of eukaryotic replication terminator proteins with the cognate termini and the replication termination mechanism. Here, we report a biochemical analysis of the interaction of the Reb1 terminator protein of Schizosaccharomyces pombe, which binds to the Ter3 site present in the nontranscribed spacers of ribosomal DNA, located in chromosome III. We show that Reb1 is a dimeric protein and that the N-terminal dimerization domain of the protein is dispensable for replication termination. Unlike its mammalian counterpart Ttf1, Reb1 did not need an accessory protein to bind to Ter3. The two myb/SANT domains and an adjacent, N-terminal 154-amino-acid-long segment (called the myb-associated domain) were both necessary and sufficient for optimal DNA binding in vitro and fork arrest in vivo. The protein and its binding site Ter3 were unable to arrest forks initiated in vivo from ars of Saccharomyces cerevisiae in the cell milieu of the latter despite the facts that the protein retained the proper affinity of binding, was located in vivo at the Ter site, and apparently was not displaced by the “sweepase” Rrm3. These observations suggest that replication fork arrest is not an intrinsic property of the Reb1-Ter3 complex.

scholarly journals Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

2016 ◽  
Vol 113 (26) ◽  
pp. E3639-E3648 ◽  
Author(s):  
Deepak Bastia ◽  
Pankaj Srivastava ◽  
Shamsu Zaman ◽  
Malay Choudhury ◽  
Bidyut K. Mohanty ◽  
...  
Keyword(s):  
Replication Fork ◽  
General Mechanism ◽  
Nonhistone Protein ◽  
In Vivo Experiments ◽  
Vitro Binding ◽  
Fork Protection Complex ◽  
Replication Fork Arrest ◽  
Replication Factors

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex.

scholarly journals Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

2021 ◽  
Author(s):  
Allison W. McClure ◽  
John F.X. Diffley
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Replication Initiation ◽  
Multiple Roles ◽  
Replication Forks ◽  
Dna Replication Stress ◽  
Necessary And Sufficient

SummaryThe Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Allison McClure ◽  
John Diffley
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Replication Initiation ◽  
Multiple Roles ◽  
Replication Forks ◽  
Dna Replication Stress ◽  
Necessary And Sufficient

The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

scholarly journals The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Liang Ge ◽  
David Melville ◽  
Min Zhang ◽  
Randy Schekman
Keyword(s):  
Autophagosome Formation ◽  
Functional Studies ◽  
A Cell ◽  
Intermediate Compartment ◽  
Membrane Isolation ◽  
Necessary And Sufficient ◽  
Organelle Membrane ◽  
Catabolic Process

Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes.

Stanniocalcin-1, an inhibitor of macrophage chemotaxis and chemokinesis

2004 ◽  
Vol 286 (2) ◽  
pp. F356-F362 ◽  
Author(s):  
John Kanellis ◽  
Roger Bick ◽  
Gabriela Garcia ◽  
Luan Truong ◽  
Chun Chui Tsao ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Intracellular Calcium ◽  
In Vivo Studies ◽  
Cellular Processes ◽  
Multiple Organs ◽  
Chemotactic Protein ◽  
Stromal Cell Derived Factor ◽  
Mammalian Counterpart

In macrophages, changes in intracellular calcium have been associated with activation of cellular processes that regulate cell adhesion and motility and are important for the response of macrophages to antigenic stimuli. The mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in multiple organs including the thymus and spleen, and hence, we hypothesized that it may have a role in modulating the immune/inflammatory response. Using murine macrophage-like (RAW264.7) and human monoblast-like (U937) cells to study chemotaxis in vitro, we found that STC1 attenuated chemokinesis and diminished the chemotactic response to monocyte chemotactic protein-1 (MCP-1) and stromal cell-derived factor-1α. Consistent with these findings, STC1 blunted the rise in intracellular calcium following MCP-1 stimulation in RAW264.7 cells. In vivo studies suggested differential expression of STC1 in obstructed kidney and localization to macrophages. MCP-1 and STC1 transcripts were both upregulated following ureteric obstruction, suggesting a functional association between the two genes. Our data suggest a role for mammalian STC1 in modulating the immune/inflammatory response.

scholarly journals ParB of Pseudomonas aeruginosa: Interactions with Its Partner ParA and Its Target parS and Specific Effects on Bacterial Growth

2004 ◽  
Vol 186 (20) ◽  
pp. 6983-6998 ◽  
Author(s):  
Aneta A. Bartosik ◽  
Krzysztof Lasocki ◽  
Jolanta Mierzejewska ◽  
Christopher M. Thomas ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Streptomyces Coelicolor ◽  
Consensus Sequence ◽  
Terminal Part ◽  
Dimerization Domain ◽  
Interaction Domain ◽  
C Terminus ◽  
Cell Components

ABSTRACT The par genes of Pseudomonas aeruginosa have been studied to increase the understanding of their mechanism of action and role in the bacterial cell. Key properties of the ParB protein have been identified and are associated with different parts of the protein. The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role. The dimerization domain of P. aeruginosa ParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group). The C-terminal part of ParB is also involved in ParB-ParA interactions. Purified ParB binds specifically to DNA containing a putative parS sequence based on the consensus sequence found in the chromosomes of Bacillus subtilis, Pseudomonas putida, and Streptomyces coelicolor. The overproduction of ParB was shown to inhibit the function of genes placed near parS. This “silencing” was dependent on the parS sequence and its orientation. The overproduction of P. aeruginosa ParB or its N-terminal part also causes inhibition of the growth of P. aeruginosa and P. putida but not Escherichia coli cells. Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus of P. aeruginosa ParB in interactions with host cell components.

scholarly journals An amphipathic helix enables septins to sense micrometer-scale membrane curvature

2019 ◽  
Vol 218 (4) ◽  
pp. 1128-1137 ◽  
Author(s):  
Kevin S. Cannon ◽  
Benjamin L. Woods ◽  
John M. Crutchley ◽  
Amy S. Gladfelter
Keyword(s):  
Membrane Curvature ◽  
Amphipathic Helix ◽  
Curvature Sensing ◽  
C Terminus ◽  
Number Of Binding Sites ◽  
Curved Membranes ◽  
Necessary And Sufficient ◽  
Micrometer Scale

Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.

Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1926-1932 ◽  
Author(s):  
Tomoaki Fujisaki ◽  
Marc G. Berger ◽  
Stefan Rose-John ◽  
Connie J. Eaves
Keyword(s):  
Growth Factors ◽  
Fold Increase ◽  
Semisolid Medium ◽  
Response Characteristics ◽  
Adult Human ◽  
Liquid Suspension ◽  
Normal Duration ◽  
Necessary And Sufficient

Abstract Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.

scholarly journals The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

Development ◽  
2020 ◽  
Vol 147 (24) ◽  
pp. dev185827
Author(s):  
Timothy Grocott ◽  
Estefania Lozano-Velasco ◽  
Gi Fay Mok ◽  
Andrea E. Münsterberg
Keyword(s):  
Model Organism ◽  
Control Gene ◽  
Tissue Interactions ◽  
Organ Systems ◽  
Necessary And Sufficient ◽  
Regenerative Therapies ◽  
Further Development ◽  
Master Control

ABSTRACTUnderstanding how complex organ systems are assembled from simple embryonic tissues is a major challenge. Across the animal kingdom a great diversity of visual organs are initiated by a ‘master control gene’ called Pax6, which is both necessary and sufficient for eye development. Yet precisely how Pax6 achieves this deeply homologous function is poorly understood. Using the chick as a model organism, we show that vertebrate Pax6 interacts with a pair of morphogen-coding genes, Tgfb2 and Fst, to form a putative Turing network, which we have computationally modelled. Computer simulations suggest that this gene network is sufficient to spontaneously polarise the developing retina, establishing the first organisational axis of the eye and prefiguring its further development. Our findings reveal how retinal self-organisation may be initiated independently of the highly ordered tissue interactions that help to assemble the eye in vivo. These results help to explain how stem cell aggregates spontaneously self-organise into functional eye-cups in vitro. We anticipate these findings will help to underpin retinal organoid technology, which holds much promise as a platform for disease modelling, drug development and regenerative therapies.

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