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Chromatin dynamics during nucleotide excision repair: histones on the move.

Adam S, Polo SE - Int J Mol Sci (2012)

Bottom Line: Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair.We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity.In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Dynamics, Curie Institute Research Centre, 75248 Paris Cedex 5, France; E-Mail: salome.adam@curie.fr ; Centre National de la Recherche Scientifique, Unité Mixte de Recherche 218, 75248 Paris Cedex 5, France.

ABSTRACT
It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.

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Related in: MedlinePlus

Methods for measuring chromatin restoration upon UVC damage. Deposition of histone proteins from a cell-free extract onto damaged DNA immobilized on magnetic beads is measured by western blotting (WB) against pulled-down histones [19,31]. Chromatin assembly coupled to NER can also be monitored in vitro by supercoiling assays using damaged plasmids mixed with extracts from human cells, xenopus eggs or drosophila embryos that are supplemented with a radioactive desoxyribonucleotide ([32P]dCTP which labels repair patches, orange). Within minutes, the plasmid is relaxed by topoisomerases present in the extracts. Nucleosome assembly introduces negative superhelical turns into the relaxed plasmid, which can be detected as faster migrating forms by electrophoresis on an agarose gel stained with Ethidium Bromide (EtBr, total DNA) or revealed by autoradiography (32P, repaired DNA) ([38,39], reviewed in [31]). In vivo, new histone deposition at sites of local UVC damage is visualized by immunofluorescence (IF) in cultured human cells transiently expressing epitope-tagged histones (e-H3.1, green) [40].
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f3-ijms-13-11895: Methods for measuring chromatin restoration upon UVC damage. Deposition of histone proteins from a cell-free extract onto damaged DNA immobilized on magnetic beads is measured by western blotting (WB) against pulled-down histones [19,31]. Chromatin assembly coupled to NER can also be monitored in vitro by supercoiling assays using damaged plasmids mixed with extracts from human cells, xenopus eggs or drosophila embryos that are supplemented with a radioactive desoxyribonucleotide ([32P]dCTP which labels repair patches, orange). Within minutes, the plasmid is relaxed by topoisomerases present in the extracts. Nucleosome assembly introduces negative superhelical turns into the relaxed plasmid, which can be detected as faster migrating forms by electrophoresis on an agarose gel stained with Ethidium Bromide (EtBr, total DNA) or revealed by autoradiography (32P, repaired DNA) ([38,39], reviewed in [31]). In vivo, new histone deposition at sites of local UVC damage is visualized by immunofluorescence (IF) in cultured human cells transiently expressing epitope-tagged histones (e-H3.1, green) [40].

Mentions: Over the past decades, a series of methods have been developed both in vitro and in vivo to assess histone and nucleosome dynamics coupled to NER (Figures 1–3), considerably increasing our understanding of chromatin rearrangements during the NER process.


Chromatin dynamics during nucleotide excision repair: histones on the move.

Adam S, Polo SE - Int J Mol Sci (2012)

Methods for measuring chromatin restoration upon UVC damage. Deposition of histone proteins from a cell-free extract onto damaged DNA immobilized on magnetic beads is measured by western blotting (WB) against pulled-down histones [19,31]. Chromatin assembly coupled to NER can also be monitored in vitro by supercoiling assays using damaged plasmids mixed with extracts from human cells, xenopus eggs or drosophila embryos that are supplemented with a radioactive desoxyribonucleotide ([32P]dCTP which labels repair patches, orange). Within minutes, the plasmid is relaxed by topoisomerases present in the extracts. Nucleosome assembly introduces negative superhelical turns into the relaxed plasmid, which can be detected as faster migrating forms by electrophoresis on an agarose gel stained with Ethidium Bromide (EtBr, total DNA) or revealed by autoradiography (32P, repaired DNA) ([38,39], reviewed in [31]). In vivo, new histone deposition at sites of local UVC damage is visualized by immunofluorescence (IF) in cultured human cells transiently expressing epitope-tagged histones (e-H3.1, green) [40].
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472782&req=5

f3-ijms-13-11895: Methods for measuring chromatin restoration upon UVC damage. Deposition of histone proteins from a cell-free extract onto damaged DNA immobilized on magnetic beads is measured by western blotting (WB) against pulled-down histones [19,31]. Chromatin assembly coupled to NER can also be monitored in vitro by supercoiling assays using damaged plasmids mixed with extracts from human cells, xenopus eggs or drosophila embryos that are supplemented with a radioactive desoxyribonucleotide ([32P]dCTP which labels repair patches, orange). Within minutes, the plasmid is relaxed by topoisomerases present in the extracts. Nucleosome assembly introduces negative superhelical turns into the relaxed plasmid, which can be detected as faster migrating forms by electrophoresis on an agarose gel stained with Ethidium Bromide (EtBr, total DNA) or revealed by autoradiography (32P, repaired DNA) ([38,39], reviewed in [31]). In vivo, new histone deposition at sites of local UVC damage is visualized by immunofluorescence (IF) in cultured human cells transiently expressing epitope-tagged histones (e-H3.1, green) [40].
Mentions: Over the past decades, a series of methods have been developed both in vitro and in vivo to assess histone and nucleosome dynamics coupled to NER (Figures 1–3), considerably increasing our understanding of chromatin rearrangements during the NER process.

Bottom Line: Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair.We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity.In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Dynamics, Curie Institute Research Centre, 75248 Paris Cedex 5, France; E-Mail: salome.adam@curie.fr ; Centre National de la Recherche Scientifique, Unité Mixte de Recherche 218, 75248 Paris Cedex 5, France.

ABSTRACT
It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair.

Show MeSH
Related in: MedlinePlus