End‐joining of reconstituted histone H2AX‐containing chromatin in vitro by soluble nuclear proteins from human cells

Non‐homologous end‐joining is an important pathway for the repair of DNA double‐strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser‐139 in the histone variant H2AX to form γ‐H2AX. Here we report efficient in vitro end‐joining of reconstituted chromatin containing nu...

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Bibliographic Details
Published in:FEBS letters 2002-09, Vol.527 (1-3), p.105-108
Main Authors: Siino, Joseph S, Nazarov, Igor B, Zalenskaya, Irina A, Yau, Peter M, Bradbury, E.Morton, Tomilin, Nikolai V
Format: Article
Language:English
Subjects:
AFM, atomic force microscopy
Androstadienes - pharmacology
Base Sequence
Cell Extracts
Cells, Cultured
Chromatin - drug effects
Chromatin - metabolism
DNA end-joining
DNA Repair - drug effects
DNA Repair - physiology
DSB, double-strand DNA break
DTT, dithiothreitol
Enzyme Inhibitors - pharmacology
Histone H2AX
Histones - drug effects
Histones - metabolism
Humans
Molecular Biology - methods
Molecular Sequence Data
NCP, nucleosome core particle
NHEJ, non-homologous end-joining
Nuclear Proteins - chemistry
Nuclear Proteins - metabolism
Phosphatidylinositol 3-Kinases - antagonists & inhibitors
Phosphorylation
Reconstituted chromatin
rhH2AX, recombinant human H2AX
Solubility
Wortmannin
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Summary:Non‐homologous end‐joining is an important pathway for the repair of DNA double‐strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser‐139 in the histone variant H2AX to form γ‐H2AX. Here we report efficient in vitro end‐joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end‐joining is not suppressed by the PI‐3 kinase inhibitor wortmannin. During the end‐joining reaction H2AX is phosphorylated at Ser‐139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. Therefore, in vitro the DNA end‐joining reaction appears to be independent of H2AX phosphorylation.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(02)03176-9