scholarly journals Ku-Dependent Nonhomologous DNA End Joining inXenopus Egg Extracts

1999 ◽  
Vol 19 (4) ◽  
pp. 2585-2593 ◽  
Author(s):  
Paul Labhart
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
End Joining ◽  
Free System ◽  
Egg Extract ◽  
Egg Extracts ◽  
Dependent Protein ◽  
Immunological Assays ◽  
Dna Ends

ABSTRACT An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907–924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5′-protruding single strand (PSS) and an end with a 3′-PSS, between two ends with 3′-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5′-PSS was Ku independent. These results show that theXenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.

scholarly journals Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis

2018 ◽  
Author(s):  
Maiko Kitaoka ◽  
Rebecca Heald ◽  
Romain Gibeaux
Keyword(s):  
Spindle Assembly ◽  
Morphometric Variation ◽  
Free System ◽  
Egg Extract ◽  
Egg Extracts ◽  
A Cell ◽  
Clawed Frog ◽  
Xenopus Borealis

ABSTRACTEgg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation.

scholarly journals A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling

2019 ◽  
Vol 47 (14) ◽  
pp. 7444-7459 ◽  
Author(s):  
Lisa Harpprecht ◽  
Sandro Baldi ◽  
Tamas Schauer ◽  
Andreas Schmidt ◽  
Tanja Bange ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
High Efficiency ◽  
Drosophila Embryo ◽  
Dna Breaks ◽  
Drosophila Cell ◽  
Free System ◽  
Cell Free System ◽  
Associated Proteins ◽  
Dna Ends

Abstract Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to γH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The γH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range γH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei.

scholarly journals XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

2015 ◽  
Vol 35 (17) ◽  
pp. 3017-3028 ◽  
Author(s):  
Sunetra Roy ◽  
Abinadabe J. de Melo ◽  
Yao Xu ◽  
Satish K. Tadi ◽  
Aurélie Négrel ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Strand Breaks ◽  
Ligase Iv ◽  
Cell Strains ◽  
Dna Ends

The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA endsin vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNAin vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.

scholarly journals The Ku80 Carboxy Terminus Stimulates Joining and Artemis-Mediated Processing of DNA Ends

2008 ◽  
Vol 29 (5) ◽  
pp. 1134-1142 ◽  
Author(s):  
Eric Weterings ◽  
Nicole S. Verkaik ◽  
Guido Keijzers ◽  
Bogdan I. Florea ◽  
Shih-Ya Wang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Nuclease Activity ◽  
Carboxy Terminus ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Repair Defect ◽  
Carboxy Terminal ◽  
Dna Ends

ABSTRACT Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and the XRCC4/ligase IV complex. Activation of the DNA-PKCS serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PKCS at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PKCS autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PKCS autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

scholarly journals CoincidentIn VitroAnalysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Cynthia L. Hendrickson ◽  
Shubhadeep Purkayastha ◽  
Elzbieta Pastwa ◽  
Ronald D. Neumann ◽  
Thomas A. Winters
Keyword(s):  
Mammalian Cells ◽  
Current Model ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Ligase Iv ◽  
Dependent Protein ◽  
Regulatory Functions

In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PKcsto form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PKcsappears to inhibit DNA-PK-independent NHEJ, andin vitroanalysis of DNA-PK-independent NHEJ in the presence of the DNA-PKcsprotein remains problematic. We have developed anin vitroassay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.

scholarly journals A noncatalytic function of the ligation complex during nonhomologous end joining

2013 ◽  
Vol 200 (2) ◽  
pp. 173-186 ◽  
Author(s):  
Jessica Cottarel ◽  
Philippe Frit ◽  
Oriane Bombarde ◽  
Bernard Salles ◽  
Aurélie Négrel ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Free System ◽  
Cell Extracts ◽  
Dna Double Strand Break ◽  
Dependent Protein ◽  
A Cell ◽  
Dna Ends

Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)–DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)–X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4–Cer-XLF complexes on DNA ends.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

scholarly journals MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts

1996 ◽  
Vol 109 (1) ◽  
pp. 239-246 ◽  
Author(s):  
A. Abrieu ◽  
T. Lorca ◽  
J.C. Labbe ◽  
N. Morin ◽  
S. Keyse ◽  
...  
Keyword(s):  
Map Kinase ◽  
Degradation Pathway ◽  
Cdc2 Kinase ◽  
Vitro Assay ◽  
Kinase Activation ◽  
Egg Extracts ◽  
Dependent Protein ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.

scholarly journals Activation of DNA-PK by hairpinned DNA ends reveals a stepwise mechanism of kinase activation

2020 ◽  
Vol 48 (16) ◽  
pp. 9098-9108 ◽  
Author(s):  
Katheryn Meek
Keyword(s):  
Protein Kinase ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Stepwise Mechanism ◽  
Dependent Protein ◽  
Enzymatic Activation ◽  
Non Homologous End Joining ◽  
Dna Ends ◽  
Step Mechanism

Abstract As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

scholarly journals Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing

2020 ◽  
Vol 21 (4) ◽  
pp. 1380 ◽  
Author(s):  
Giovanni Pasquini ◽  
Virginia Cora ◽  
Anka Swiersy ◽  
Kevin Achberger ◽  
Lena Antkowiak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Time Course ◽  
Sequencing Analysis ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Gene ◽  
Repair Pathways

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

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