Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate - Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair
Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate - Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair
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DOI：
10.1074/jbc.m102125200
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发表时间：
2001-07-06
影响因子：
4.8
通讯作者：
Wilson, SH
中科院分区：
文献类型：
--
作者：
Lavrik, OI;Prasad, R;Wilson, SH
为了研究哺乳动物碱基切除修复(BER)酶与BER过程中形成的DNA中间体的相互作用，我们使用了一种新型的光亲和标记探针和小鼠胚胎成纤维细胞的细胞提取物。该探针是利用含有合成碱性位点的末端标记的寡核苷酸原位形成的；该位点被无嘌呤/脱嘧啶核酸内切酶切割，在边缘形成具有3‘-羟基和5’-还原糖磷酸基团的缺口，然后将携带光反应加合物的dNMP添加到3‘-羟基。用近紫外光(312 Nm)照射萃取物/探针混合物，6个蛋白质被强标记。其中包括聚腺苷二磷酸核糖聚合酶-1(PARP-1)和BER参与者Flat核酸内切酶-1、DNA聚合酶β和脱嘌呤/脱嘧啶核酸内切酶，与PARP-1交联量大于与其他蛋白质交联量。用不同竞争对手的寡核苷酸和具有不同结构的DNA探针检测PARP-1标记的特异性。PARP-1在代表BER中间体的DNA上的标记比在双链DNA中代表缺口的标记更强，这些结果表明可以从细胞粗提物中筛选出优先与光反应性BER中间体相互作用的蛋白质。
To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3 ' -hydroxyl and 5 ' -reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3 ' -hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease, The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA, These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.