Luo C M, Tang W, Mekeel K L, DeFrank J S, Anné P R, Powell S N
Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
J Biol Chem. 1996 Feb 23;271(8):4497-503. doi: 10.1074/jbc.271.8.4497.
The only specific DNA repair defect found in ataxia telangiectasia (A-T) cells is mis-repair of cleaved DNA. In this report we measured DNA recombination, given its role in DNA repair and genetic instability. Using plasmids containing selectable reporter genes, we found a higher frequency of both chromosomal recombination (>100 times) and extra-chromosomal recombination (27 times) in SV40-transformed A-T cell lines compared with in an SV40-transformed normal fibroblast cell line. Southern analysis of single A-T colonies exhibiting post-integration recombination revealed that 24/27 had undergone aberrant rearrangements; recombination in normal fibroblast colonies was achieved by gene conversion in 8/11 clones and 10/11 clones showed unchanged copies of the plasmid. Using co-transfection of two integrating plasmids, each containing a separate deletion in the xgprt reporter gene, the 27 times difference in extra-chromosomal recombination was found when the plasmids were cleaved at a distance from the reporter gene. When the plasmids were cleaved within the reporter gene, the co-transfection frequency was reduced in A-T, but was increased in normal cells. We conclude that A-T cell lines have not only a high frequency chromosomal and extra-chromosomal recombination, but also exhibit error-prone recombination of cleaved DNA.
共济失调毛细血管扩张症（A-T）细胞中发现的唯一特异性DNA修复缺陷是切割后DNA的错配修复。在本报告中，鉴于DNA重组在DNA修复和基因不稳定中的作用，我们对其进行了测量。使用含有可选择报告基因的质粒，我们发现与SV40转化的正常成纤维细胞系相比，SV40转化的A-T细胞系中染色体重组（>100倍）和染色体外重组（27倍）的频率更高。对表现出整合后重组的单个A-T菌落进行Southern分析显示，24/27发生了异常重排；正常成纤维细胞菌落中的重组通过8/11个克隆中的基因转换实现，10/11个克隆中的质粒拷贝未发生变化。使用共转染两个整合质粒，每个质粒在xgprt报告基因中含有一个单独的缺失，当质粒在距报告基因一定距离处切割时，发现染色体外重组存在27倍的差异。当质粒在报告基因内切割时，A-T中的共转染频率降低，但在正常细胞中增加。我们得出结论，A-T细胞系不仅具有高频的染色体和染色体外重组，而且还表现出切割后DNA的易错重组。
