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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 disorganization upon UVC damage. Spontaneous changes in DNA folding in reconstituted nucleosomes containing UV photoproducts (red triangles) is measured by FRET (Fluorescence Resonance Energy Transfer) between the donor (Cy3) and acceptor (Cy5) fluorophores [21]. Chromatin rearrangements upon UVC damage can also be assessed by probing the accessibility of damaged DNA to nucleases (orange scissors), which is performed either on reconstituted chromatin (in vitro) or on chromatin purified from damaged cells (in vivo). The accumulation of small DNA fragments, indicative of disorganized chromatin (by nucleosome disruption and/or random positioning), is visualized by electrophoresis on an agarose gel stained with Ethidium Bromide (EtBr) [28,29,31]. In vivo, changes in histone density can be visualized by a decrease in fluorescent signal at sites of local UVC damage in cultured human cells expressing GFP-tagged histones (green) [32].
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f2-ijms-13-11895: Methods for measuring chromatin disorganization upon UVC damage. Spontaneous changes in DNA folding in reconstituted nucleosomes containing UV photoproducts (red triangles) is measured by FRET (Fluorescence Resonance Energy Transfer) between the donor (Cy3) and acceptor (Cy5) fluorophores [21]. Chromatin rearrangements upon UVC damage can also be assessed by probing the accessibility of damaged DNA to nucleases (orange scissors), which is performed either on reconstituted chromatin (in vitro) or on chromatin purified from damaged cells (in vivo). The accumulation of small DNA fragments, indicative of disorganized chromatin (by nucleosome disruption and/or random positioning), is visualized by electrophoresis on an agarose gel stained with Ethidium Bromide (EtBr) [28,29,31]. In vivo, changes in histone density can be visualized by a decrease in fluorescent signal at sites of local UVC damage in cultured human cells expressing GFP-tagged histones (green) [32].

Mentions: Early work studying how the NER pathway repairs UVC lesions on naked DNA revealed that, despite the short size of the repair patch (about 30 nucleotides in length) [26], access to at least 100 base pairs flanking the lesion was needed for the repair machinery to excise the damaged oligonucleotide [27]. These early observations suggested that, in vivo, the chromatin structure should be rearranged in order for repair factors to process UVC lesions. Formal proof of chromatin reorganization upon DNA damage was obtained by measuring chromatin accessibility to nucleases, which preferentially cleave the DNA between nucleosome particles (Figure 2). Pioneering experiments using partial MNase (Micrococcal Nuclease) digestion on chromatin purified from UVC-irradiated human fibroblasts [28] showed that chromatin regions undergoing NER present a transient increase in nuclease sensitivity. These initial observations were further confirmed by using other nucleases such as DNase I (DNA Nuclease 1) [29] or restriction enzymes [30].


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

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

Methods for measuring chromatin disorganization upon UVC damage. Spontaneous changes in DNA folding in reconstituted nucleosomes containing UV photoproducts (red triangles) is measured by FRET (Fluorescence Resonance Energy Transfer) between the donor (Cy3) and acceptor (Cy5) fluorophores [21]. Chromatin rearrangements upon UVC damage can also be assessed by probing the accessibility of damaged DNA to nucleases (orange scissors), which is performed either on reconstituted chromatin (in vitro) or on chromatin purified from damaged cells (in vivo). The accumulation of small DNA fragments, indicative of disorganized chromatin (by nucleosome disruption and/or random positioning), is visualized by electrophoresis on an agarose gel stained with Ethidium Bromide (EtBr) [28,29,31]. In vivo, changes in histone density can be visualized by a decrease in fluorescent signal at sites of local UVC damage in cultured human cells expressing GFP-tagged histones (green) [32].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ijms-13-11895: Methods for measuring chromatin disorganization upon UVC damage. Spontaneous changes in DNA folding in reconstituted nucleosomes containing UV photoproducts (red triangles) is measured by FRET (Fluorescence Resonance Energy Transfer) between the donor (Cy3) and acceptor (Cy5) fluorophores [21]. Chromatin rearrangements upon UVC damage can also be assessed by probing the accessibility of damaged DNA to nucleases (orange scissors), which is performed either on reconstituted chromatin (in vitro) or on chromatin purified from damaged cells (in vivo). The accumulation of small DNA fragments, indicative of disorganized chromatin (by nucleosome disruption and/or random positioning), is visualized by electrophoresis on an agarose gel stained with Ethidium Bromide (EtBr) [28,29,31]. In vivo, changes in histone density can be visualized by a decrease in fluorescent signal at sites of local UVC damage in cultured human cells expressing GFP-tagged histones (green) [32].
Mentions: Early work studying how the NER pathway repairs UVC lesions on naked DNA revealed that, despite the short size of the repair patch (about 30 nucleotides in length) [26], access to at least 100 base pairs flanking the lesion was needed for the repair machinery to excise the damaged oligonucleotide [27]. These early observations suggested that, in vivo, the chromatin structure should be rearranged in order for repair factors to process UVC lesions. Formal proof of chromatin reorganization upon DNA damage was obtained by measuring chromatin accessibility to nucleases, which preferentially cleave the DNA between nucleosome particles (Figure 2). Pioneering experiments using partial MNase (Micrococcal Nuclease) digestion on chromatin purified from UVC-irradiated human fibroblasts [28] showed that chromatin regions undergoing NER present a transient increase in nuclease sensitivity. These initial observations were further confirmed by using other nucleases such as DNase I (DNA Nuclease 1) [29] or restriction enzymes [30].

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