CHARACTERIZATION OF POLYMERS OF ADENOSINE-DIPHOSPHATE RIBOSE GENERATED INVITRO AND INVIVO
CHARACTERIZATION OF POLYMERS OF ADENOSINE-DIPHOSPHATE RIBOSE GENERATED INVITRO AND INVIVO
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DOI：
10.1021/bi00385a042
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发表时间：
1987-06-02
期刊：
影响因子：
2.9
通讯作者：
JACOBSON, MK
中科院分区：
文献类型：
--
作者：
ALVAREZGONZALEZ, R;JACOBSON, MK
已经开发并应用了方法来确定在核苷酸可渗透培养的小鼠细胞和在完整的培养细胞中合成的ADP-核糖聚合物的大小和分支频率。用硼树脂亲和层析纯化聚合物，并用分子筛高效液相色谱按大小分级。将各馏分用酶法消化成核苷酸，用强阴离子交换高效液相色谱分离。根据这些数据，计算了聚合物的平均尺寸和支化频率。观察到了大范围的聚合物尺寸。在体外产生了长达190个残基的聚合物，每个分子至少有5个支化点。在DNA烷化剂N-甲基-N‘-硝基-N-亚硝基处理后，从完整的细胞中分离出最多67个残基的聚合物，每个分子最多包含两个支化点。在DNA损伤之前经过高温处理的细胞含有平均最大尺寸为244个残基的聚合物，每个分子最多包含6个分支点。在完整细胞中检测到大的ADP-核糖聚合物表明，多(ADP-核糖化)对染色质组织的影响可能超出共价修饰蛋白质的范围，很可能涉及多(ADP-核糖)与染色质的其他成分的非共价相互作用。
Methods have been developed and applied to determine the size and branching frequency of polymers of ADP-ribose synthesized in nucleotide-permeable cultured mouse cells and in intact cultured cells. Polymers were purified by affinity chromatography with a boronate resin and were fractionated according to size by molecular sieve high-performance liquid chromatography. Fractions were enzymatically digested to nucleotides, which were separated by strong anion exchange high-performance liquid chromatography. From these data, average polymer size and branching frequency were calculated. A wide range of polymer sizes was observed. Polymers as large as 190 residues with at least five points of branching per molecule were generated in vitro. Polymers of up to 67 residues containing up to two points of branching per molecule were isolated from intact cells following treatment with the DNA alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine. Cells treated with hyperthermia prior to DNA damage contained polymers of an average maximum size of 244 residues containing up to six points of branching per molecule. The detection of large polymers of ADP-ribose in intact cells suggests that alterations in chromatin organization effected by poly(ADP-ribosylation) may extend beyond the covalently modified proteins and very likely involve noncovalent interactions of poly(ADP-ribose) with other components of chromatin.