Pietrowska Monika, Widłak Piotr
Department of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland.
Acta Biochim Pol. 2005;52(4):867-74. Epub 2005 Jun 6.
Proteins recognizing DNA damaged by the chemical carcinogen N-acetoxy-acetylaminofluorene (AAAF) were analyzed in nuclear extracts from rat tissues, using a 36 bp oligonucleotide as a substrate and electrophoretic mobility shift and Southwestern blot assays. One major damage-recognizing protein was detected, whose amount was estimated as at least 10(5) copies per cell. Levels of this protein were similar in extracts from brain, kidney and liver, but much lower in extracts from testis. The affinity of the detected protein for DNA damaged by AAAF was about 70-fold higher than for undamaged DNA. DNA damaged by cis-diamminedichloroplatinum (cis-DDP), benzo(a)pyrene diolepoxide (BPDE) or UV-radiation also bound this protein with an increased affinity, the former more strongly and the latter two more weakly as compared to AAAF-damaged DNA. The detected AAAF/DDP-damaged-DNA-binding (AAAF/DDP-DDB) protein had a molecular mass of about 25 kDa and was distinct from histone H1 or HMGB proteins, which are known to have a high affinity for cis-DDP-damaged DNA. The level of this damage-recognizing protein was not affected in rats treated with the carcinogen 2-acetylaminofluorene. The activity of an AAAF/DDP-DDB protein could also be detected in extracts from mouse liver cells but not from the Hep2G human hepatocellular carcinoma.
利用一段36bp的寡核苷酸作为底物，通过电泳迁移率变动分析和蛋白质免疫印迹分析，对大鼠组织核提取物中识别被化学致癌物N-乙酰氧基-乙酰氨基芴（AAAF）损伤的DNA的蛋白质进行了分析。检测到一种主要的损伤识别蛋白，其含量估计为每个细胞至少10⁵个拷贝。该蛋白在脑、肾和肝的提取物中的水平相似，但在睾丸提取物中的水平要低得多。检测到的蛋白对被AAAF损伤的DNA的亲和力比对未损伤DNA的亲和力高约70倍。被顺式二氨二氯铂（cis-DDP）、苯并(a)芘二环氧物（BPDE）或紫外线辐射损伤的DNA也能以增加的亲和力与该蛋白结合，与被AAAF损伤的DNA相比，前者结合更强，后两者结合较弱。检测到的AAAF/顺铂损伤DNA结合（AAAF/DDP-DDB）蛋白的分子量约为25 kDa，与已知对顺铂损伤DNA具有高亲和力的组蛋白H1或HMGB蛋白不同。在用致癌物2-乙酰氨基芴处理的大鼠中，这种损伤识别蛋白的水平没有受到影响。在小鼠肝细胞提取物中也能检测到AAAF/DDP-DDB蛋白的活性，但在人肝癌细胞系Hep2G的提取物中未检测到。
