Calsou P, Salles B
Laboratoire de Pharmacologie et Toxicologie Fondamentales du CNRS, Toulouse-France.
Biochem Biophys Res Commun. 1994 Jul 29;202(2):788-95. doi: 10.1006/bbrc.1994.1999.
We have devised a method to evaluate the capacity of mammalian cell extracts to incise damaged DNA in vitro. The assay uses damaged-plasmid DNA as a substrate for nucleotide excision repair by cell extracts. During this process, enzymatic incision of the damaged DNA is followed by DNA resynthesis. Under our assay conditions, the DNA synthesis stage of excision repair is prevented by limiting dNTP concentration and including the specific DNA polymerase inhibitor aphidicolin. Incisions are quantitatively detected by [alpha-32P]dAMP incorporation catalysed by the Klenow fragment of E. coli DNA pol I at nicked sites in plasmids purified from incision reactions. Lesion-specific incision is an ATP-dependent process; it was observed in plasmids modified with three different DNA damaging agents and damage-dependent incisions were abolished with extracts from xeroderma pigmentosum excision-repair deficient cell lines, indicating that this in vitro incision assay is dealing with true nucleotide excision repair.
我们设计了一种方法来评估哺乳动物细胞提取物在体外切割受损DNA的能力。该检测方法使用受损质粒DNA作为细胞提取物进行核苷酸切除修复的底物。在此过程中，受损DNA的酶切之后是DNA再合成。在我们的检测条件下，通过限制dNTP浓度并加入特定的DNA聚合酶抑制剂阿非迪霉素来阻止切除修复的DNA合成阶段。通过在从切割反应中纯化的质粒的切口位点处，由大肠杆菌DNA聚合酶I的Klenow片段催化的[α-32P]dAMP掺入来定量检测切口。损伤特异性切口是一个ATP依赖的过程；在用三种不同的DNA损伤剂修饰的质粒中观察到了这一过程，并且来自色素性干皮病切除修复缺陷细胞系的提取物消除了损伤依赖性切口，这表明这种体外切口检测方法针对的是真正的核苷酸切除修复。
