Single-strand breakage in mammalian chromosomal DNA: sensitive detection by application of a sedimentation anomaly.
Single-strand breakage in mammalian chromosomal DNA: sensitive detection by application of a sedimentation anomaly.
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哺乳动物染色体 DNA 中的单链断裂：通过应用沉降异常进行灵敏检测。
DOI：
10.1080/09553008014550711
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
M. Hartwig
中科院分区：
文献类型：
--
作者：
M. Hartwig
真核染色体DNA在中性蔗糖梯度中的沉降研究不仅丰富了我们对染色质高阶结构的认识，而且为检测DNA单链断裂提供了新的方法。从各种真核细胞温和释放到中性蔗糖梯度中的染色体 DNA 已被证明折叠成环，其中旋转受限的 DNA 呈超螺旋状。该结果是使用离子型（Ide、Nakane、Anzai 和 Andoh 1975、Hartwig 1977、1978；Nakane、Ide、Anzai、Ohara 和 Andoh 1978）和非离子型（Cook 和 Brazell 1975、1976 a、Benyajati 和 Worcel 1976、Pinon 和 Salts 1977）洗涤剂获得的。在裂解过程中。染色体 DNA 中的单链断裂被认为会降低其沉降速率 (1) 由于单环内超螺旋的释放 (Cook 和 Brazell 1975、Benyajati 和 Worcel 1976、Hartwig 1977、Nakane 等人 1978) 和 (2) 由于 DNA 解折叠 (Hartwig 1978)。后一过程的分子机制目前尚不清楚，尽管可能表明单链断裂影响 DNA 与非组蛋白的结合，而非组蛋白似乎参与维持真核染色体 DNA 的环结构（Ide 等人，1975 年，Hartwig 1977 年，Pinon 和 Salts 1977 年）。在本通讯中，表明，在离子去污剂十二烷基硫酸钠 (SDS) 中进行细胞裂解的情况下，当应用转子速度效应时，可以通过其在中性蔗糖梯度中降低的沉降速率来灵敏地检测到哺乳动物染色体 DNA 中的单链断裂。中国仓鼠细胞（V79/4 系）在 SDS 中的裂解释放出与非组蛋白和脂质复合物内的染色体 DNA，但已耗尽组蛋白（下文中称为“简单复合体”）。如果仅以 4 x 10 3 rpm 进行离心，则 DNA 单链断裂作用的目标大小（根据细胞 X 辐射时复合物沉降速率下降 63% 推断得出）位于 DNA 环大小的范围内，以排除沉降异常（Hartwig 1978）。然而，在对小鼠肿瘤细胞进行 X 射线照射时，当以 30 x 103 rpm 的转速进行离心时，DNA 单链断裂的目标似乎要大两个数量级（Drasil、Karpfel、Juraskova 和 Ryznar 1972）。因此，DNA单链断裂引起的沉降速率的变化可能取决于离心速度。事实证明这个期望是正确的。中国仓鼠细胞释放的复合物的沉淀条件已有描述（Hartwig 1978）。简而言之，将 0. 1 ml 含有约 5 x 104 个细胞的悬浮液分层到 4.6 ml 5-20 细胞上方的 0. 2 ml 裂解介质（1% SDS、0. 2% 脱氧胆酸钠、0. 05 M EDTA、0.05 M 柠檬酸钠）上。
Sedimentation studies of eukaryotic chromosomal DNA in neutral sucrose gradients have not only enriched our knowledge on higher-order structure of chromatin, but have provided a new method for detecting DNA single-strand breakage. Chromosomal DNA released gently from a variety of eukaryotic cells into neutral sucrose gradients has been shown to be folded into loops in which the rotationally restricted DNA is supercoiled. This result has been obtained using both ionic (Ide, Nakane, Anzai and Andoh 1975, Hartwig 1977, 1978; Nakane, Ide, Anzai, Ohara and Andoh 1978) and non-ionic (Cook and Brazell 1975, 1976 a, Benyajati and Worcel 1976, Pinon and Salts 1977) detergents in the lysis procedure. Single-strand breakage in chromosomal DNA has been proposed to decrease its sedimentation rate (1) due to release of supercoiling within single loops (Cook and Brazell 1975, Benyajati and Worcel 1976, Hartwig 1977, Nakane et al. 1978) and (2) due to unfolding of DNA (Hartwig 1978). The molecular mechanism of the latter process is not clear at present, although it may be suggested that single-strand breakage affects the binding of DNA to non-histone proteins which seem to be involved in maintaining the loop structure in eukaryotic chromosomal DNA (Ide et al. 1975, Hartwig 1977, Pinon and Salts 1977). In this communication it is shown that, in the case of cell lysis in the ionic detergent sodium dodecyl sulphate (SDS), single-strand breakage in mammalian chromosomal DNA can be sensitively detected by its decreased sedimentation rate in neutral sucrose gradients when applying a rotor speed effect.Lysis of Chinese hamster cells (line V79/4) in SDS releases the chromosomal DNA within a complex with non-histone proteins and lipids, but depleted of histones (called simplycomplex'in the following). The target size for the action of DNA single-strand breakage, as deduced from a 63 per cent decline in the sedimentation rate of the complex on X-irradiation of the cells, is in the range of the DNA loop size if centrifugation is performed at 4 x 10 3 rpm only, in order to exclude sedimentation anomalies (Hartwig 1978). However, on X-irradiation of mouse tumour cells the target for DNA single-strand breakage seems to be about two orders of magnitude larger when centrifugation is performed at 30 x 103 rpm (Drasil, Karpfel, Juraskova and Ryznar 1972). Therefore, the change in sedimentation rate induced by DNA single-strand breakage might be dependent on the speed of centrifugation. This expectation proved to be true. The conditions for sedimentation of the complex released from Chinese hamster cells have been described (Hartwig 1978). Briefly, 0. 1 ml of a suspension, containing about 5 x 104 cells, was layered onto 0. 2 ml of the lysis medium (1 per cent SDS, 0. 2 per cent Na-deoxycholate, 0. 05 M EDTA, 0.05 M Na-citrate) above a 4.6 ml 5-20