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

• 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
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(02)03176-9

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.