scholarly journals Recombination occurs within minutes of replication blockage by RTS1 producing restarted forks that are prone to collapse

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Michael O Nguyen ◽  
Manisha Jalan ◽  
Carl A Morrow ◽  
Fekret Osman ◽  
Matthew C Whitby
Keyword(s):  
Cell Cycle ◽  
Replication Fork ◽  
Genome Duplication ◽  
Time Lapse ◽  
Genetic Change ◽  
Replication Proteins ◽  
Site Specific ◽  
Time Lapse Microscopy ◽  
A Site ◽  
Stalled Fork

The completion of genome duplication during the cell cycle is threatened by the presence of replication fork barriers (RFBs). Following collision with a RFB, replication proteins can dissociate from the stalled fork (fork collapse) rendering it incapable of further DNA synthesis unless recombination intervenes to restart replication. We use time-lapse microscopy and genetic assays to show that recombination is initiated within ∼10 min of replication fork blockage at a site-specific barrier in fission yeast, leading to a restarted fork within ∼60 min, which is only prevented/curtailed by the arrival of the opposing replication fork. The restarted fork is susceptible to further collapse causing hyper-recombination downstream of the barrier. Surprisingly, in our system fork restart is unnecessary for maintaining cell viability. Seemingly, the risk of failing to complete replication prior to mitosis is sufficient to warrant the induction of recombination even though it can cause deleterious genetic change.

scholarly journals Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

2009 ◽  
Vol 29 (17) ◽  
pp. 4742-4756 ◽  
Author(s):  
Alexander Lorenz ◽  
Fekret Osman ◽  
Victoria Folkyte ◽  
Sevil Sofueva ◽  
Matthew C. Whitby
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Replication Fork ◽  
Direct Repeat ◽  
Dna Helicase ◽  
Replication Forks ◽  
Site Specific ◽  
Replicative Stress ◽  
Recombinant Formation ◽  
A Site

ABSTRACT Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.

scholarly journals Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis

1998 ◽  
Vol 28 (5) ◽  
pp. 883-892 ◽  
Author(s):  
Chris D. Webb ◽  
Peter L. Graumann ◽  
Jason A. Kahana ◽  
Aurelio A. Teleman ◽  
Pamela A. Silver ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bacillus Subtilis ◽  
Replication Origin ◽  
Time Lapse ◽  
Time Lapse Microscopy ◽  
Rapid Movement ◽  
Use Of Time ◽  
Origin Region

scholarly journals In-phase oscillation of global regulons is orchestrated by a pole-specific organizer

2016 ◽  
Vol 113 (44) ◽  
pp. 12550-12555 ◽  
Author(s):  
Balaganesh Janakiraman ◽  
Johann Mignolet ◽  
Sharath Narayanan ◽  
Patrick H. Viollier ◽  
Sunish Kumar Radhakrishnan
Keyword(s):  
Cell Cycle ◽  
Cell Fate ◽  
Caulobacter Crescentus ◽  
Transcriptional Regulators ◽  
Forward Genetics ◽  
Cell Fate Determination ◽  
Global Regulator ◽  
Site Specific ◽  
Phase Oscillation ◽  
A Site

Cell fate determination in the asymmetric bacteriumCaulobacter crescentus(Caulobacter) is triggered by the localization of the developmental regulator SpmX to the old (stalked) cell pole during the G1→S transition. Although SpmX is required to localize and activate the cell fate-determining kinase DivJ at the stalked pole inCaulobacter, in cousins such asAsticcacaulis, SpmX directs organelle (stalk) positioning and possibly other functions. We define the conserved σ54-dependent transcriptional activator TacA as a global regulator inCaulobacterwhose activation by phosphorylation is indirectly down-regulated by SpmX. Using a combination of forward genetics and cytological screening, we uncover a previously uncharacterized and polarized component (SpmY) of the TacA phosphorylation control system, and we show that SpmY function and localization are conserved. Thus, SpmX organizes a site-specific, ancestral, and multifunctional regulatory hub integrating the in-phase oscillation of two global transcriptional regulators, CtrA (the master cell cycle transcriptional regulator A) and TacA, that perform important cell cycle functions.

scholarly journals Microtubules in Candida albicans Hyphae Drive Nuclear Dynamics and Connect Cell Cycle Progression to Morphogenesis

2005 ◽  
Vol 4 (10) ◽  
pp. 1697-1711 ◽  
Author(s):  
Kenneth R. Finley ◽  
Judith Berman
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Basal Cell ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Microtubule Dynamics ◽  
Growth Defect ◽  
Cell Cortex ◽  
Long Distance ◽  
Time Lapse Microscopy

ABSTRACT Candida albicans is an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicans hyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 μm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicans nuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.

scholarly journals Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death

2018 ◽  
Author(s):  
Zeynep Kabakci ◽  
Simon Käppeli ◽  
Giorgio Cozza ◽  
Claudio Cantù ◽  
Christiane König ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mixed Type ◽  
Molecular Level ◽  
Time Lapse ◽  
Guided Discovery ◽  
Enzymatic Assays ◽  
Intestinal Crypt ◽  
Time Lapse Microscopy ◽  
Cycle Arrest ◽  
Mechanism Of Inhibition

ABSTRACTCDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

DNA synthesis errors associated with double-strand-break repair.

Genetics ◽  
1995 ◽  
Vol 140 (3) ◽  
pp. 965-972 ◽  
Author(s):  
J N Strathern ◽  
B K Shafer ◽  
C B McGill
Keyword(s):  
Dna Synthesis ◽  
Dna Cleavage ◽  
Genome Duplication ◽  
Inducible Promoter ◽  
Strand Break ◽  
Site Specific ◽  
Large Populations ◽  
Dna Break ◽  
A Site ◽  
Ho Gene

Abstract Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele. Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene. Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells. To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1. The reversion rate of the trp1 alleles was approximately 100-fold higher among cells that had experienced an HO cut than among uninduced cells. The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage.

scholarly journals Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Guillaume Witz ◽  
Erik van Nimwegen ◽  
Thomas Julou
Keyword(s):  
Cell Cycle ◽  
Time Lapse ◽  
Growth Conditions ◽  
Replication Initiation ◽  
Stochastic Simulations ◽  
E Coli ◽  
Time Lapse Microscopy ◽  
Statistical Framework ◽  
Wide Range ◽  
Cell Cycle Models

Living cells proliferate by completing and coordinating two cycles, a division cycle controlling cell size and a DNA replication cycle controlling the number of chromosomal copies. It remains unclear how bacteria such as Escherichia coli tightly coordinate those two cycles across a wide range of growth conditions. Here, we used time-lapse microscopy in combination with microfluidics to measure growth, division and replication in single E. coli cells in both slow and fast growth conditions. To compare different phenomenological cell cycle models, we introduce a statistical framework assessing their ability to capture the correlation structure observed in the data. In combination with stochastic simulations, our data indicate that the cell cycle is driven from one initiation event to the next rather than from birth to division and is controlled by two adder mechanisms: the added volume since the last initiation event determines the timing of both the next division and replication initiation events.

Time‐Lapse Microscopy Approaches to Track Cell Cycle and Lineage Progression at the Single‐Cell Level

2013 ◽  
Vol 64 (1) ◽  
Author(s):  
Rachel J. Errington ◽  
Sally C. Chappell ◽  
Imtiaz A. Khan ◽  
Nuria Marquez ◽  
Marie Wiltshire ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Time Lapse ◽  
Single Cell Level ◽  
Cell Level ◽  
Time Lapse Microscopy

scholarly journals Aurora A site specific TACC3 phosphorylation regulates astral microtubule assembly by stabilizing γ-tubulin ring complex

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Resmi Rajeev ◽  
Puja Singh ◽  
Ananya Asmita ◽  
Ushma Anand ◽  
Tapas K. Manna
Keyword(s):  
Molecular Mechanisms ◽  
Coiled Coil ◽  
Time Lapse ◽  
Microtubule Assembly ◽  
Aurora A ◽  
Site Specific ◽  
Spindle Poles ◽  
Ring Complex ◽  
Division Axis ◽  
A Site

Abstract Background Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. Results Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components. Conclusions The results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.

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