ATM protein purified from vaccinia virus expression system: DNA binding requirements for kinase activation

被引：6

作者：

Chun, HH

Cary, RB

Lansigan, F

Whitelegge, J

Rawlings, DJ

Gatti, RA ^{[1
]}

机构：

[1] Univ Calif Los Angeles, David Geffen Sch Med, Dept Pathol, Los Angeles, CA 90095 USA

[2] Los Alamos Natl Lab, Biosci Div, Los Alamos, NM 87545 USA

[3] Univ Calif Los Angeles, David Geffen Sch med, Dept Pediat, Los Angeles, CA 90095 USA

[4] Univ Calif Los Angeles, Inst Neuropsychiat, Dept Psychiat & Biobehav Sci, Pasarow Mass Spectrometry Lab, Los Angeles, CA 90095 USA

[5] Univ Calif Los Angeles, Inst Neuropsychiat, Dept Chem & Biochem, Pasarow Mass Spectrometry Lab, Los Angeles, CA 90095 USA

[6] Univ Washington, Sch Med, Dept Pediat, Seattle, WA 98195 USA

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2004年 / 322卷 / 01期

关键词：

ataxia-telangiectasia; ATM; vaccinia virus; purification; expression; protein kinase; atomic force microscopy; DNA; recombinant protein;

D O I：

10.1016/j.bbrc.2004.07.085

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The ataxia-telangiectasia, mutated (ATM) gene product plays a role in responding to double stand DNA breaks. Some biochemical studies of ATM function have been hampered by lack of an efficient expression system and abundant purified ATM protein. We report the construction of a vaccinia virus expressing ATM, vWR-ATM, which was used to produce large amounts of functional FLAG-tagged ATM protein (FLAG-ATM) in HeLa cells. Kinase activity of the purified FLAG-ATM was dependent on manganese and inhibited with wortmannin. Using the FLAG-ATM recombinant protein, GST-p53 serine 15 phosphorylation increased in the presence of damaged DNA. PHAS-1 phosphorylation was found to be DNA independent. Purified FLAG-ATM was recovered in the autophosphorylated form, as demonstrated by phosphorylation of ATM serine 1981. As shown by atomic force microscopy, FLAG-ATM bound to linear DNA both at broken ends and in mid-strands. Vaccinia virus is the most efficient ATM expression system described to date. (C) 2004 Elsevier Inc. All rights reserved.

引用

页码：74 / 81

页数：8

共 31 条

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