Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2.
Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2.
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DOI：
10.1016/s0021-9258(18)42242-9
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发表时间：
1992-07
期刊：
影响因子：
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通讯作者：
Edward N. Hughes;B. N. Engelsberg;Paul C. Billings
中科院分区：
文献类型：
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作者：
Edward N. Hughes;B. N. Engelsberg;Paul C. Billings
在人类细胞中负责识别和修复顺铂损伤的DNA的生物化学过程还没有得到很好的理解。我们已经开发了一种受损的DNA亲和沉淀技术，允许直接可视化和表征的细胞蛋白结合顺铂损伤的DNA。该方法从复杂的放射性标记的细胞混合物中分离受损的DNA结合蛋白，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳将其进一步分解为单个多肽。这种技术是互补的凝胶阻滞和西南印迹分析，以前已被用来确定细胞成分，特异性结合顺铂损伤的DNA。使用这种技术，我们已经确定了一组HeLaS 3核蛋白的26.5，28，90，和97 kDa的特异性结合顺铂-DNA加合物。可溶性顺铂损伤DNA的竞争研究证实了这些发现。26.5和28 kDa的主要顺铂损伤的DNA结合蛋白识别与顺铂修饰的DNA加合物，但不与其反式异构体或与UV辐射。通过离子交换和顺铂损伤的DNA亲和层析将这些蛋白质纯化450倍至接近同质。氨基末端序列分析表明，26.5-和28-kDa的蛋白是相同的高迁移率族（HMG）蛋白HMG-2和HMG-1，分别。
The biochemical processes responsible for the recognition and repair of cisplatin-damaged DNA in human cells are not well understood. We have developed a damaged DNA affinity precipitation technique that allows the direct visualization and characterization of cellular proteins that bind to cisplatin-damaged DNA. The method separates damaged DNA-binding proteins from complex radiolabeled cell mixtures and further resolves them into individual polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique is complementary to gel retardation and Southwestern blotting analyses that have been previously used to identify cellular components that specifically bind to cisplatin-damaged DNA. Using this technique, we have characterized a set of HeLaS3 nuclear proteins of 26.5, 28, 90, and 97 kDa that specifically bind to cisplatin-DNA adducts. Competition studies with soluble cisplatin-damaged DNA confirmed these findings. The major cisplatin-damaged DNA-binding proteins of 26.5 and 28 kDa recognized adducts of DNA modified with cisplatin but not with its trans-isomer or with UV radiation. These proteins were purified 450-fold to near homogeneity by ion-exchange and cisplatin-damaged DNA affinity chromatography. Amino-terminal sequence analysis showed that the 26.5- and 28-kDa proteins were identical to high mobility group (HMG) proteins HMG-2 and HMG-1, respectively.