scholarly journals Heterochromatin is refractory to γ-H2AX modification in yeast and mammals

2007 ◽  
Vol 178 (2) ◽  
pp. 209-218 ◽  
Author(s):  
Jung-Ae Kim ◽  
Michael Kruhlak ◽  
Farokh Dotiwala ◽  
André Nussenzweig ◽  
James E. Haber
Keyword(s):  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Strand Break ◽  
Ataxia Telangiectasia Mutated ◽  
Exogenous Dna ◽  
Histone H2ax ◽  
Mouse Embryo Fibroblasts ◽  
Yeast Chromosome ◽  
High Level ◽  
Chromosomal Sequences

Double-strand break (DSB) damage in yeast and mammalian cells induces the rapid ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia and Rad3 related)-dependent phosphorylation of histone H2AX (γ-H2AX). In budding yeast, a single endonuclease-induced DSB triggers γ-H2AX modification of 50 kb on either side of the DSB. The extent of γ-H2AX spreading does not depend on the chromosomal sequences. DNA resection after DSB formation causes the slow, progressive loss of γ-H2AX from single-stranded DNA and, after several hours, the Mec1 (ATR)-dependent spreading of γ-H2AX to more distant regions. Heterochromatic sequences are only weakly modified upon insertion of a 3-kb silent HMR locus into a γ-H2AX–covered region. The presence of heterochromatin does not stop the phosphorylation of chromatin more distant from the DSB. In mouse embryo fibroblasts, γ-H2AX distribution shows that γ-H2AX foci increase in size as chromatin becomes more accessible. In yeast, we see a high level of constitutive γ-H2AX in telomere regions in the absence of any exogenous DNA damage, suggesting that yeast chromosome ends are transiently detected as DSBs.

scholarly journals Programmed phosphorylation of histone H2AX precedes a phase of DNA double-strand break-independent synapsis in mouse meiosis

Reproduction ◽  
2012 ◽  
Vol 144 (6) ◽  
pp. 699-712 ◽  
Author(s):  
Josefa Blanco-Rodríguez
Keyword(s):  
Chromosome Segregation ◽  
Ataxia Telangiectasia ◽  
Strand Break ◽  
Ataxia Telangiectasia Mutated ◽  
Dsb Repair ◽  
Late Zygotene ◽  
Histone H2ax ◽  
Dna Double Strand Break ◽  
Specific Step ◽  
Second Wave

Accurate homologue synapsis during meiosis is essential for faithful chromosome segregation and formation of viable gametes. The finding ofSpo11-dependent gamma-H2AX (γH2AX) formation during leptotene and data on mutant mice have led to the notion that synapsis in mammals depends on meiotic DNA double-stranded break (DSB) repair. A second wave of ataxia telangiectasia mutated (ATM) and Rad3-related (ATR)-dependent γH2AX formation has been observed inAtm-null mice during zygotene, suggesting that this wave of phosphorylation also occurs in normal mice. Here I aimed to confirm and to analyse in deep this wave of phosphorylation. Immunostaining of spread spermatocytes shows that γH2AX accumulates on the short last axis stretches to pair. This accumulation appears within all the nuclei undergoing a specific step of late zygotene and disappears from every spermatocyte immediately after pairing completion. This γH2AX signal co-localises with ATR, isSpo11-independent and does not co-localise with free DNA 3′-end labelling. I conclude that ATR/γH2AX asynapsis signalling at the end of zygotene belongs to a physiologically programmed pathway operating at a specific meiotic step, and I propose that this pathway is involved in the triggering of a phase of DSB-independent chromosome pairing that leads to synapsis completion in normal mouse meiosis.

scholarly journals Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

2011 ◽  
Vol 286 (22) ◽  
pp. 19229-19236 ◽  
Author(s):  
Laura A. Lindsey-Boltz ◽  
Aziz Sancar
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Effector Proteins ◽  
Ataxia Telangiectasia Mutated ◽  
Checkpoint Kinase ◽  
Atr Kinase

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

ATR (ataxia telangiectasia mutated- and Rad3-related kinase) is activated by mild hypothermia in mammalian cells and subsequently activates p53

2011 ◽  
Vol 435 (2) ◽  
pp. 499-508 ◽  
Author(s):  
Anne Roobol ◽  
Jo Roobol ◽  
Martin J. Carden ◽  
Amandine Bastide ◽  
Anne E. Willis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Mild Hypothermia ◽  
Chinese Hamster ◽  
Membrane Lipid ◽  
Mitogen Activated Protein Kinase ◽  
Ataxia Telangiectasia Mutated ◽  
Mitogen Activated Protein

In vitro cultured mammalian cells respond to mild hypothermia (27–33 °C) by attenuating cellular processes and slowing and arresting the cell cycle. The slowing of the cell cycle at the upper range (31–33 °C) and its complete arrest at the lower range (27–28 °C) of mild hypothermia is effected by the activation of p53 and subsequent expression of p21. However, the mechanism by which cold is perceived in mammalian cells with the subsequent activation of p53 has remained undetermined. In the present paper, we report that the exposure of Chinese-hamster ovary-K1 cells to mildly hypothermic conditions activates the ATR (ataxia telangiectasia mutated- and Rad3-related kinase)–p53–p21 signalling pathway and is thus a key pathway involved in p53 activation upon mild hypothermia. In addition, we show that although p38MAPK (p38 mitogen-activated protein kinase) is also involved in activation of p53 upon mild hypothermia, this is probably the result of activation of p38MAPK by ATR. Furthermore, we show that cold-induced changes in cell membrane lipid composition are correlated with the activation of the ATR–p53–p21 pathway. Therefore we provide the first mechanistic detail of cell sensing and signalling upon mild hypothermia in mammalian cells leading to p53 and p21 activation, which is known to lead to cell cycle arrest.

scholarly journals Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events

1994 ◽  
Vol 14 (2) ◽  
pp. 1293-1301
Author(s):  
K M Kramer ◽  
J A Brock ◽  
K Bloom ◽  
J K Moore ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Site ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
High Level ◽  
A Site ◽  
Mat Locus ◽  
Mechanical Rupture

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.

Strand Invasion and DNA Synthesis From the Two 3′ Ends of a Double-Strand Break in Mammalian Cells

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1439-1447 ◽  
Author(s):  
Richard D McCulloch ◽  
Leah R Read ◽  
Mark D Baker
Keyword(s):  
Dna Synthesis ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromosomal Locus ◽  
Vector Integration ◽  
Vector Borne ◽  
Strand Invasion ◽  
Chromosomal Sequences ◽  
Targeting Vector

AbstractAnalysis of the crossover products recovered following transformation of mammalian cells with a sequence insertion (“ends-in”) gene-targeting vector revealed a novel class of recombinant. In this class of recombinants, a single vector copy has integrated into an ectopic genomic position, leaving the structure of the cognate chromosomal locus unaltered. Thus, in this respect, the recombinants resemble simple cases of random vector integration. However, the important difference is that the two paired 3′ vector ends have acquired endogenous, chromosomal sequences flanking both sides of the vector-borne double-strand break (DSB). In some cases, copying was extensive, extending >16 kb into nonhomologous flanking DNA. The results suggest that mammalian homologous recombination events can involve strand invasion and DNA synthesis by both 3′ ends of the DSB. These DNA interactions are a central, predicted feature of the DSBR model of recombination.

scholarly journals Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

1994 ◽  
Vol 14 (2) ◽  
pp. 1293-1301 ◽  
Author(s):  
K M Kramer ◽  
J A Brock ◽  
K Bloom ◽  
J K Moore ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Site ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
High Level ◽  
A Site ◽  
Mat Locus ◽  
Mechanical Rupture

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.

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