Deletion mutants of poly(ADP-ribose) polymerase support a model of cyclic association and dissociation of enzyme from DNA ends during DNA repair.
Deletion mutants of poly(ADP-ribose) polymerase support a model of cyclic association and dissociation of enzyme from DNA ends during DNA repair.
复制标题
聚（ADP-核糖）聚合酶的缺失突变体支持 DNA 修复过程中酶从 DNA 末端循环缔合和解离的模型。
DOI：
10.1021/bi00186a018
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Cherney,B
中科院分区：
文献类型：
--
作者：
Smulson,M;Istock,N;Ding,R;Cherney,B
材料和方法细胞培养.从美国国立卫生研究院的Norman库珀处获得适于旋转培养的HeLa细胞，并将其添加到补充有5%（v/v）马血清（Gibco-BRL）的MEM旋转培养基（Quality Biologicals）中。HeLa细胞提取物根据Manley等人的方案制备。（1983年）。用于试验性实验的全细胞提取物是来自美国国立卫生研究院的约翰布雷迪和迈克尔拉多诺维奇的礼物。通过使提取物通过双链DNA-纤维素（Sigma）柱来耗尽PADPRP（Satoh & Lindahl，1992）。用反义RNA构建体转染的HeLa细胞（Ding等人，1992）在150-mm培养皿中生长至接近汇合，并通过刮取收获。如前所述，用1 mM地塞米松诱导它们的PADPRP反义表达72小时（Ding等人，1992年）。DNA修复反应7-如前所述制备在DNA修复反应中用作底物的经辐照的pBluescript质粒（Satoh & Lindahl，1992）。简言之，将CsCl纯化的闭环质粒在γ辐射器100中暴露于来自137 Cs的50戈伊γ射线。完全或PADPRP耗尽的提取物在细胞提取物重悬缓冲液中稀释至所需的蛋白质浓度，如Manley等人所述。（1983年）。适当时，将重组PADPRP蛋白加入到PADPRP耗尽的提取物中，并包括在加入反应混合物的提取物体积中。将NAD（终浓度2 mM）以溶解在20 mM Hepes（pH 7.8）中的IOmM储备溶液的形式加入到反应混合物中。肌酸
MATERIALS AND METHODSCell Culture. HeLa cells adapted for spinner culture were obtained from Norman Cooper at the National Institutes of Health and maintainedin MEM Spinner Medium (Quality Biologicals) supplemented with 5%(v/v) horse serum (Gibco-BRL). HeLa cell extracts were prepared according to the protocol of Manley et al.(1983). Whole-cell extracts for pilot experiments were a gift from John Brady and Michael Radonovich at the National Institutes of Health. Extracts were depleted of PADPRP by passage through a column of double-stranded DNA-cellulose (Sigma)(Satoh & Lindahl, 1992). HeLa cells transfected with an antisense RNA construct (Ding et al., 1992) were grown in 150-mm dishes to near confluence and harvested by scraping. They were induced for PADPRP antisense expression for 72 h with 1 mM dexamethasone as described in detail earlier (Ding et al., 1992). DNA Repair Reactions. 7-Irradiated pBluescript plasmids for use as substrate in the DNA repair reactions were prepared as previously described (Satoh & Lindahl, 1992). Briefly, CsCl-purified closed circular plasmids were exposed to 50 Gy of 7-rays from 137Cs in a Gamma Radiator 100. Complete or PADPRP-depleted extracts were diluted to thedesired protein concentration in cell extract resuspension buffer described by Manley et al.(1983). When appropriate, recombinant PADPRP proteins were added to PADPRP-depleted extract and includedin the volume of extract added to the reactionmixture. NAD (final concentration 2 mM) was added to the reaction mixture in the form of a 10 mM stock solution dissolved in 20 mM Hepes (pH 7.8). Creatine