Dresler S L, Roberts J D, Lieberman M W
Biochemistry. 1982 May 11;21(10):2557-64. doi: 10.1021/bi00539a040.
We have extended our permeable cell system for measuring DNA excision repair [Roberts, J. D., & Lieberman, M. W. (1979) Biochemistry 18, 4499-4505] so that steps of the repair process, beginning with incision and extending at least through the "rearrangement" of repaired nucleosomes which follows repair synthesis, all take place in permeable cells. In the revised protocol, human fibroblasts are made permeable, damaged with UV or chemicals in suspension, and incubated with a reaction mix containing ATP and the four deoxyribonucleoside triphosphates, one of which is labeled with 32P. By reducing the exogenous dNTP concentration to 3 microM and including 15 mM KCl in the reaction mixture, we have greatly reduced background incorporation in undamaged cells without significantly reducing repair synthesis. This permits us to measure repair synthesis without separating it from replicative synthesis by isopycnic centrifugation. Repair synthesis in this system is very similar to that occurring in intact cells: in response to DNA damage, nucleotides are incorporated into DNA of parental density (when analyzed by the BrdUrd density shift technique), incorporation increases with increasing DNA damage, synthesis is dependent on the presence of all four dNTPs, and the system accurately reflects the genetic UV repair deficiency of xeroderma pigmentosum (XP) cells. Furthermore, as has been observed in intact cells, repair-incorporated nucleotides in these permeable cells are initially overrepresented in staphylococcal nuclease sensitive regions of chromatin and are subsequently redistributed to give a nearly uniform distribution between nuclease-sensitive and -resistant regions. The UV dose curve of permeable cells differs somewhat from that of intact cells; however, the dose differs somewhat from that of intact cells; however, the dose curve for permeable cells treated with N-methyl-N-nitrosourea is very similar to that of intact cells. Repair synthesis in UV-damaged, permeable normal and XP cells is stimulated by addition of Micrococcus luteus UV endonuclease, indicating that the damaged DNA is accessible to exogenous repair enzymes and suggesting that incision, or an obligatory preincision step, is rate limiting for excision repair in these permeable cells. Repair synthesis in this system is inhibited by aphidicolin, but not by high levels of dideoxy-TTP, suggesting involvement of DNA polymerase alpha in excision repair. Novobiocin is also inhibitory alpha and the HeLa cell type II DNA topoisomerase.
我们扩展了用于测量DNA切除修复的可渗透细胞系统[罗伯茨，J.D.，&利伯曼，M.W.（1979年）《生物化学》18卷，4499 - 4505页]，以使修复过程的各个步骤，从切口开始，至少延伸到修复合成后修复核小体的“重排”，都能在可渗透细胞中发生。在修订后的方案中，使人成纤维细胞具有渗透性，在悬浮液中用紫外线或化学物质损伤，然后与含有ATP和四种脱氧核糖核苷三磷酸的反应混合物一起孵育，其中一种用32P标记。通过将外源dNTP浓度降低到3 microM，并在反应混合物中加入15 mM KCl，我们大大降低了未受损细胞中的背景掺入，而不会显著降低修复合成。这使我们能够测量修复合成，而无需通过等密度离心将其与复制合成分离。该系统中的修复合成与完整细胞中的非常相似：响应DNA损伤，核苷酸掺入亲本密度的DNA中（通过BrdUrd密度转移技术分析时），掺入随着DNA损伤的增加而增加，合成依赖于所有四种dNTP的存在，并且该系统准确反映了着色性干皮病（XP）细胞的遗传性紫外线修复缺陷。此外，正如在完整细胞中观察到的那样，这些可渗透细胞中修复掺入的核苷酸最初在染色质的葡萄球菌核酸酶敏感区域中占比过高，随后重新分布，在核酸酶敏感和抗性区域之间给出几乎均匀的分布。可渗透细胞的紫外线剂量曲线与完整细胞的有所不同；然而，剂量与完整细胞的有所不同；然而，用N - 甲基 - N - 亚硝基脲处理的可渗透细胞的剂量曲线与完整细胞的非常相似。添加藤黄微球菌紫外线内切酶可刺激紫外线损伤的、可渗透的正常细胞和XP细胞中的修复合成，这表明受损DNA对外源修复酶是可及的，并表明切口或一个必需的切口前步骤是这些可渗透细胞中切除修复的限速步骤。该系统中的修复合成受到阿非科林的抑制，但不受高水平双脱氧TTP的抑制，这表明DNA聚合酶α参与了切除修复。新生霉素也抑制α和HeLa细胞II型DNA拓扑异构酶。
