Brois D W, McMahon L W, Ramos N I, Anglin L M, Walsh C E, Lambert M W
Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA.
Carcinogenesis. 1999 Sep;20(9):1845-53. doi: 10.1093/carcin/20.9.1845.
Cells from individuals with the cancer-prone, inherited disorder Fanconi anemia (FA) are hypersensitive to DNA interstrand cross-linking agents and this hypersensitivity correlates with a defect in ability to repair this type of damage to their DNA. We have isolated a DNA endonuclease complex from the nuclei of normal human cells which is involved in repair of DNA interstrand cross-links and have shown that in FA complementation group A (FA-A) cells there is a defect in ability of this complex to incise DNA containing interstrand cross-links. In order to identify the specific protein(s) in this complex which is defective in FA-A cells, monoclonal antibodies (mAbs) were developed against proteins in the normal complex. One of these mAbs, which is against a protein with a molecular weight of approximately 230 kDa, completely inhibited the ability of the normal complex to incise cross-linked DNA. Western blot analysis has shown that there is a deficiency in this protein in FA-A cells. Electophoretic analysis has also indicated that there are reduced levels of this protein in FA-A compared with normal cells. Studies carried out utilizing FA-A cells which have been stably transduced with a retroviral vector expressing the FANCA cDNA have shown that the DNA repair defect in these cells has been corrected; levels of unscheduled DNA synthesis are at least as great as those of normal human cells. In addition, in the transduced cells the deficiency in the 230 kDa protein has been corrected, as determined by both western blot and electrophoretic analysis. These results indicate that the FANCA gene plays a role in the expression or stability of the 230 kDa protein.
患有易患癌症的遗传性疾病范可尼贫血（FA）的个体的细胞对DNA链间交联剂高度敏感，这种超敏反应与修复这类DNA损伤的能力缺陷相关。我们从正常人细胞的细胞核中分离出一种参与DNA链间交联修复的DNA内切酶复合物，并表明在FA互补组A（FA-A）细胞中，该复合物切割含有链间交联的DNA的能力存在缺陷。为了鉴定该复合物中在FA-A细胞中有缺陷的特定蛋白质，针对正常复合物中的蛋白质制备了单克隆抗体（mAb）。其中一种针对分子量约为230 kDa的蛋白质的mAb完全抑制了正常复合物切割交联DNA的能力。蛋白质印迹分析表明FA-A细胞中这种蛋白质存在缺陷。电泳分析也表明，与正常细胞相比，FA-A细胞中这种蛋白质的水平降低。利用用表达FANCA cDNA的逆转录病毒载体稳定转导的FA-A细胞进行的研究表明，这些细胞中的DNA修复缺陷已得到纠正；非定标DNA合成水平至少与正常人细胞一样高。此外，通过蛋白质印迹和电泳分析确定，在转导的细胞中，230 kDa蛋白质的缺陷已得到纠正。这些结果表明FANCA基因在230 kDa蛋白质的表达或稳定性中起作用。
