Topologically constrained domains of supercoiled DNA in eukaryotic cells.
Topologically constrained domains of supercoiled DNA in eukaryotic cells.
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真核细胞中超螺旋 DNA 的拓扑约束结构域。
DOI：
10.1089/dna.1997.16.1051
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Vaughan,AT
中科院分区：
文献类型：
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作者：
Khodarev,NN;Narayana,A;Constantinou,A;Vaughan,AT
通过与一组辐照过的超螺旋质粒DNA直接比较，确定了鳞状细胞癌细胞系SQ-20 B内超螺旋、拓扑结构受限的DNA结构域的大小。通过凝胶电泳和类核流式细胞术测定质粒和细胞中超螺旋的损失。质粒松弛的剂量-反应数据与从SQ-20 B细胞获得的数据的比较使得能够直接估计这些细胞中的超螺旋靶尺寸。使用质粒pUCD 9 P（3.9 kbp）、pXT-1（10.1 kbp）、pdBPV-MMT-neo（14.6 kbp）、pRK 290（20.0 kbp）和R6 K（38 kbp），并在与类核DNA相同的暴露条件下进行分析。在类核中发现了两种大小的拓扑闭合结构域，大小分别为0.51 ± 0.17Mbp和1.34 ± 0.3Mbp。为了将这些大规模的DNA组织与功能联系起来，将细胞暴露于DNA拓扑异构酶II抑制剂VP 16和G1/S细胞周期阻断剂含羞草素。暴露于VP 16 1 h可有效降低DNA合成，这与类核超螺旋平行增加有关。添加G1> S抑制剂含羞草素增强了这两种反应。它的结论是，染色体和间期核组织成至少两个大小的拓扑约束域的DNA可能具有功能相关的控制和执行的DNA合成。
The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comparison with a panel of irradiated supercoiled plasmid DNAs. Loss of supercoiling in plasmids was determined by gel electrophoresis and in cells by nucleoid flow cytometry. Comparison of dose-response data for plasmid relaxation with that obtained from SQ-20B cells enabled a direct estimation of supercoil target size in these cells. Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT-neo (14.6 kbp), pRK290 (20.0 kbp), and R6K (38 kbp) were used and analyzed under the same exposure conditions as nucleoid DNA. Two sizes of topologically closed domains were found in nucleoids of 0.51 ± 0.17Mbp and 1.34 ± 0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with function, cells were exposed to the DNA topoisomerase II inhibitor, VP16 and the G1/S cell cycle blocking agent mimosine. A 1 h exposure to VP16 was effective in reducing DNA synthesis which was associated with a parallel increase in nucleoid supercoiling. Addition of the G1> S inhibitor mimosine enhanced both responses. It is concluded that chromosomes and interphase nuclei are organized into at least two sizes of topologically constrained domains of DNA which may have functional relevance to the control and execution of DNA synthesis.