A human endonuclease activity for gamma-irradiated DNA.
A human endonuclease activity for gamma-irradiated DNA.
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对伽马射线照射的 DNA 具有人类核酸内切酶活性。
DOI：
10.1016/s0006-3495(73)85993-4
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发表时间：
1973
影响因子：
3.4
通讯作者：
T. Brent
中科院分区：
文献类型：
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作者：
T. Brent
在微生物中，有令人信服的证据表明DNA修复过程有助于从辐射损伤中恢复（Howard-Flanders，1968年）。尤其是切除修复系统已在分子、酶和遗传水平上得到广泛表征（Setlow和Setlow，1972）。虽然在哺乳动物细胞中已经发现了切除系统的生化证据，但其功能意义主要来自与微生物的类比。唯一的例外是着色性干皮病（XP），这是一种遗传性人类皮肤病，其中细胞对紫外线过敏，并且缺乏切除修复的生化反应（Cleaver，1968，1971）。到目前为止，还没有一种参与哺乳动物切除修复的酶被确定。最近在HeLa细胞中发现了作用于UV照射的DNA的内切核酸酶活性（Bac-chetti et al.，1972;布伦特，1972）和XP细胞（Bacchetti等，1972年）。由于XP细胞中的功能缺陷似乎是在UV诱导的嘧啶二聚体位点处的初始核酸内切切口（Cleaver，1971），因此体外测定的人核酸内切酶活性不执行该功能。这一结论得到了以下观察结果的加强，即HeLa细胞的核酸内切活性在紫外线照射的DNA的光复活后持续存在（Bacchetti et al.，1972年）。在所观察到的核酸内切酶活性可以作用于不同于UV二聚体的类型的多种损伤，并且这种损伤可以由UV以外的辐射模式产生的前提下，我检查了γ-辐射的DNA对核酸内切酶活性的敏感性。酶提取物通过在10 mM Tris-磷酸盐缓冲液（pH 8）中超声处理HeLa细胞，并在100 ℃下加热来制备，将PM2 DNA溶解于100 mM NaCl溶液、1 mM EDTA、20 mM Tris-HCl，pH 7中，浓度为约100 μ g/ml，在室温下用60 Co源的γ射线以330 rad/min的剂量率照射PM2 DNA，通过将PM2 DNA与HeLa细胞孵育来测定核酸内切酶活性在1 mM EDTA存在下在pH 7下提取，随后通过在碱性蔗糖梯度上的速度沉降将超螺旋DNA底物与带切口的DNA产物分离（布伦特，1972）。根据相应放射性峰下每分钟的相对计数计算酶活性。在超螺旋和切口DNA峰下回收梯度上的所有放射性。切口DNA峰的对称形状表明在这些实验中不存在广泛的核酸外切酶作用。在一个典型的实验中，3,300拉德的伽马射线将大约50%的
There is compelling evidence, in microorganisms, for DNA repair processes that contribute to recovery from radiation damage (Howard-Flanders, 1968). The ex-cision repair system in particular has been extensively characterized at the molecular, enzymatic, and genetic levels (Setlow and Setlow, 1972). Although biochemical evidence for the excision system has been found in mammalian cells, its functional significance derives largely from analogy with microorganisms. The one exception is xeroderma pigmentosum (XP), a hereditary human skin disease in which cells are hypersensitive to UV light and deficient in the biochemical reactions of ex-cision repair (Cleaver, 1968, 1971). So far none of the enzymes involved inmammalian excision repair have been positively identified. An endonuclease activity which acts on UV-irradiated DNA has recently been found in HeLa cells (Bac-chetti et al., 1972; Brent, 1972) and in XP cells (Bacchetti et al., 1972). Since the function defective in XP cells appears to be an initial endonucleolytic incision at the site of UV-induced pyrimidine dimers (Cleaver, 1971), it follows that the human endonuclease activity assayed in vitro does not perform this function. This con-clusion is reinforced by the observation that HeLa cell endonucleolytic activity persists after photoreactivation of UV-irradiated DNA (Bacchetti et al., 1972). On the premise that the observed endonuclease activity may act on a variety of lesions of a type distinct from UV dimers and that such lesions can be produced by modes of radiation other than UV, I examined gamma-irradiated DNA for susceptibility to endonuclease activity.The enzyme extract was prepared by sonicating HeLa cells in 10 mM Tris-phosphate buffer, pH 8, and centrifuging at 100,000 g for 30 min. PM2 DNA, dissolved in 100 mM NaCl solution, 1 mM EDTA, 20 mM Tris-HCl, pH 7, at a con-centration of about 100, ug per ml, was irradiated under airat room temperature with gamma rays from a 60Co source at a dose rate of 330 rad per min. Endonuclease activity was assayed by incubating the PM2 DNA with the HeLa cell extract at pH 7 in the presence of 1 mM EDTA and subsequently separating the superhelical DNA substrate from the nicked DNA product by velocity sedimentation on alkaline sucrose gradients (Brent, 1972). Enzyme activity was calculated from the relative counts per minute under the respective peaks of radioactivity. All radioactivity on the gradients was recovered under the superhelical and nicked DNA peaks. The symmetrical shape of the nicked DNA peaks indicated theabsence of extensive exonuclease action in these experiments. In a typical experiment, 3,300 rad of gamma rays converted about 50% of the