Szymkowski D E, Hajibagheri M A, Wood R D
Imperial Cancer Research Fund, Clare Hall Laboratories South Mimms, Herts, U.K.
J Mol Biol. 1993 May 20;231(2):251-60. doi: 10.1006/jmbi.1993.1280.
To characterize the process by which the mammalian nucleotide excision repair complex interacts with DNA to recognize and repair lesions, we have investigated the size and distribution of repair patches induced by human cell extracts in ultraviolet light-irradiated plasmid DNA. Repair synthesis was carried out in a buffer substituting biotinylated dUTP for dTTP, to allow repair patches to be detected by electron microscopy after streptavidin/colloidal gold labelling. Individual repair events on circular plasmids that had undergone repair synthesis in cell extracts were scored as gold particles bound specifically to irradiated molecules. Samples of over 2000 irradiated and unirradiated plasmids were counted. Repair synthesis at ultraviolet light photoproducts typically replaced about 30 nucleotides, since 69% of patches contained only one particle of 10 nm gold and 24% of patches contained two gold particles (each covering approx. 29 nucleotides). In addition, the ordering of repair events among damaged plasmids closely fitted a Poisson distribution, indicating that repair of lesions is achieved via a non-processive, random diffusion mechanism. This suggests that the repair complex is not intrinsically processive.
为了描述哺乳动物核苷酸切除修复复合体与DNA相互作用以识别和修复损伤的过程，我们研究了人细胞提取物在紫外线照射的质粒DNA中诱导的修复补丁的大小和分布。修复合成在一种用生物素化的dUTP替代dTTP的缓冲液中进行，以便在链霉亲和素/胶体金标记后通过电子显微镜检测修复补丁。对在细胞提取物中进行了修复合成的环状质粒上的单个修复事件进行评分，将其作为与辐照分子特异性结合的金颗粒。对超过2000个辐照和未辐照的质粒样本进行了计数。紫外线光产物处的修复合成通常替换约30个核苷酸，因为69%的补丁仅包含一个10纳米金颗粒，24%的补丁包含两个金颗粒（每个覆盖约29个核苷酸）。此外，受损质粒之间修复事件的排序非常符合泊松分布，表明损伤的修复是通过非连续的随机扩散机制实现的。这表明修复复合体本质上不是连续的。
