scholarly journals Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

2020 ◽  
Vol 117 (35) ◽  
pp. 21354-21363 ◽  
Author(s):  
Kevin Li ◽  
Gabriel Bronk ◽  
Jane Kondev ◽  
James E. Haber
Keyword(s):  
Three Dimensional ◽  
Strand Break ◽  
Double Strand Break ◽  
Three Dimensions ◽  
Histone H2a ◽  
Directed Motion ◽  
Checkpoint Kinases ◽  
Dna Double Strand Break ◽  
Chromosome Iii ◽  
A Site

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeastSaccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATRor Tel1ATMcheckpoint kinases. We induced a site-specific DSB with HO endonuclease at theMATlocus on chromosome III and monitored the formation of γ-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1ATRand Tel1ATMpropagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin intrans—at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

scholarly journals Yeast ATM and ATR use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

2019 ◽  
Author(s):  
Kevin Li ◽  
Gabriel Bronk ◽  
Jane Kondev ◽  
James E. Haber
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Three Dimensions ◽  
One Dimension ◽  
Histone H2a ◽  
Directed Motion ◽  
Checkpoint Kinases ◽  
Dna Double Strand Break ◽  
Chromosome Iii ◽  
A Site

AbstractOne of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeast S. cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATR or Tel1ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by ChIP-qPCR in order to uncover the mechanisms by which Mec1ATR and Tel1ATM propagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans – at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a 3D diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

scholarly journals Gene recombination in X-ray-sensitive hamster cells.

1987 ◽  
Vol 7 (4) ◽  
pp. 1409-1414 ◽  
Author(s):  
A A Hamilton ◽  
J Thacker
Keyword(s):  
Chinese Hamster ◽  
Cell Types ◽  
Strand Break ◽  
Double Strand Break ◽  
Transformation Frequency ◽  
Homologous Region ◽  
X Ray ◽  
Dna Concentration ◽  
Dna Double Strand Break ◽  
A Site

Recombination was measured in Chinese hamster ovary (CHO-K1) cells and in the X-ray-sensitive mutants xrs1 and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the different cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-K1 and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-K1, but variation in the data was considerable. A much larger difference between CHO-K1 and the xrs mutants was found when the DNA concentration dependence of transformation was explored. While the transformation frequency of CHO-K1 increased linearly with DNA concentration, no such increase occurred with the xrs mutants irrespective of whether complete plasmids or pairs of deletion plasmids were transferred. The fraction of cells taking up DNA, assayed autoradiographically, was similar in all cell types. Therefore we suggest that while homologous recombination of plasmid molecules may not be substantially reduced in the xrs mutants,processes involved in the stable integration of plasmid DNA into genomic DNA are significantly impaired.

scholarly journals Molecular Analysis of Base Damage Clustering Associated with a Site-Specific Radiation-Induced DNA Double-Strand Break

2006 ◽  
Vol 166 (5) ◽  
pp. 767-781 ◽  
Author(s):  
Kamal Datta ◽  
Pawel Jaruga ◽  
Miral Dizdaroglu ◽  
Ronald D. Neumann ◽  
Thomas A. Winters
Keyword(s):  
Molecular Analysis ◽  
Strand Break ◽  
Double Strand Break ◽  
Base Damage ◽  
Site Specific ◽  
Dna Double Strand Break ◽  
Radiation Induced ◽  
A Site

Gene recombination in X-ray-sensitive hamster cells

1987 ◽  
Vol 7 (4) ◽  
pp. 1409-1414
Author(s):  
A A Hamilton ◽  
J Thacker
Keyword(s):  
Chinese Hamster ◽  
Cell Types ◽  
Strand Break ◽  
Double Strand Break ◽  
Transformation Frequency ◽  
Homologous Region ◽  
X Ray ◽  
Dna Concentration ◽  
Dna Double Strand Break ◽  
A Site

Recombination was measured in Chinese hamster ovary (CHO-K1) cells and in the X-ray-sensitive mutants xrs1 and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the different cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-K1 and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-K1, but variation in the data was considerable. A much larger difference between CHO-K1 and the xrs mutants was found when the DNA concentration dependence of transformation was explored. While the transformation frequency of CHO-K1 increased linearly with DNA concentration, no such increase occurred with the xrs mutants irrespective of whether complete plasmids or pairs of deletion plasmids were transferred. The fraction of cells taking up DNA, assayed autoradiographically, was similar in all cell types. Therefore we suggest that while homologous recombination of plasmid molecules may not be substantially reduced in the xrs mutants,processes involved in the stable integration of plasmid DNA into genomic DNA are significantly impaired.

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