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

Joseph S. Siino; Igor B. Nazarov; Irina A. Zalenskaya; Peter M. Yau; E. Morton Bradbury; Nikolai V. Tomilin

doi:10.1016/S0014-5793(02)03176-9

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

Joseph S. Siino, Igor B. Nazarov, Irina A. Zalenskaya, Peter M. Yau, E. Morton Bradbury, Nikolai V. Tomilin

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English (US)
Pages (from-to)	105-108
Number of pages	4
Journal	FEBS Letters
Volume	527
Issue number	1-3
DOIs	https://doi.org/10.1016/S0014-5793(02)03176-9
State	Published - Sep 11 2002
Externally published	Yes

Keywords

DNA end-joining
Histone H2AX
Phosphorylation
Reconstituted chromatin

ASJC Scopus subject areas

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

Online availability

10.1016/S0014-5793(02)03176-9

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title = "End-joining of reconstituted histone H2AX-containing chromatin in vitro by soluble nuclear proteins from human cells",

abstract = "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.",

keywords = "DNA end-joining, Histone H2AX, Phosphorylation, Reconstituted chromatin",

author = "Siino, {Joseph S.} and Nazarov, {Igor B.} and Zalenskaya, {Irina A.} and Yau, {Peter M.} and Bradbury, {E. Morton} and Tomilin, {Nikolai V.}",

note = "Funding Information: This work is supported by the US Department of Energy Grant DE-FG03-01ER63070 and by the Russian Fund for Basic Research Grant 01-04-49486. ",

year = "2002",

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doi = "10.1016/S0014-5793(02)03176-9",

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T1 - End-joining of reconstituted histone H2AX-containing chromatin in vitro by soluble nuclear proteins from human cells

AU - Siino, Joseph S.

AU - Nazarov, Igor B.

AU - Zalenskaya, Irina A.

AU - Yau, Peter M.

AU - Bradbury, E. Morton

AU - Tomilin, Nikolai V.

N1 - Funding Information: This work is supported by the US Department of Energy Grant DE-FG03-01ER63070 and by the Russian Fund for Basic Research Grant 01-04-49486.

PY - 2002/9/11

Y1 - 2002/9/11

N2 - 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.

AB - 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.

KW - DNA end-joining

KW - Histone H2AX

KW - Phosphorylation

KW - Reconstituted chromatin

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ER -

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

Abstract

Keywords

ASJC Scopus subject areas

Online availability

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