How damaged is the biologically active subpopulation of transfected DNA?
How damaged is the biologically active subpopulation of transfected DNA?
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转染 DNA 的生物活性亚群受损程度如何？
DOI：
10.1128/mcb.4.3.387-398.1984
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发表时间：
1984
影响因子：
5.3
通讯作者：
Wilson,JH
中科院分区：
文献类型：
--
作者：
Wake,CT;Gudewicz,T;Porter,T;White,A;Wilson,JH
对于转染的DNA分子在从细胞外进入细胞核的过程中所遭受的损伤，我们知之甚少。为了选择性地跟踪完成这一旅程的次要亚群，我们设计了一种遗传方法，使用用DEAE-葡聚糖转染的猿猴病毒40 DNA。我们用三种方法研究了这个活跃的亚群：（i）通过测定仅在两个突变基因组的排列上不同的突变线性二聚体的相互对，（ii）通过测定一系列长度为1.1至2.0个猿猴病毒40个基因组的野生型寡聚体，（iii）通过测定一系列长度为1.1至2.0个猿猴病毒40个基因组的野生型寡聚体，（iv）通过测定一系列长度为1.1至2.0个猿猴病毒40个基因组的野生型寡聚体，（iv）通过测定一系列长度为1.1至2.0个猿猴病毒40个基因组的野生型寡聚体，（iv）通过测定一系列长度为1.1至2.0个猿猴病毒40个基因组的野生型寡聚体。和（iii）通过测定在非必需区域内被切割以留下粘性、平端或错配末端的猿猴病毒40的线性单体。我们从这些研究中得出结论，转染的DNA分子在活跃的亚群中受到中度损伤的片段化和修饰的结束。作为一个整体，活性亚群每5至15个内切酶遭受约一个断裂，并且约15至20%的分子的一端或两端被修饰。我们对片段化的分析与随机引入的双链断裂一致，其原因和确切性质尚不清楚。我们对末端修饰的分析表明，最普遍的损伤形式涉及少于25个碱基对的缺失或添加。此外，我们直接证明了连接粘性，钝性或错配末端的效率是相同的，验证了细胞连接几乎任何两个DNA末端的明显能力，并表明连接效率接近100%。这些实验的设计确保了检测到的损伤先于病毒复制，因此应该是所有用DEAE-葡聚糖转染的DNA所共有的，而不是病毒DNA特异性的。这些测量转染的DNA内的损伤有重要的后果，在体细胞中的同源和非同源重组的研究进行了讨论。
Relatively little is known about the damage suffered by transfected DNA molecules during their journey from outside the cell into the nucleus. To follow selectively the minor subpopulation that completes this journey, we devised a genetic approach using simian virus 40 DNA transfected with DEAE-dextran. We investigated this active subpopulation in three ways: (i) by assaying reciprocal pairs of mutant linear dimers which differed only in the arrangement of two mutant genomes; (ii) by assaying a series of wild-type oligomers which ranged from 1.1 to 2.0 simian virus 40 genomes in length; and (iii) by assaying linear monomers of simian virus 40 which were cleaved within a nonessential region to leave either sticky, blunt, or mismatched ends. We conclude from these studies that transfected DNA molecules in the active subpopulation are moderately damaged by fragmentation and modification of ends. As a whole, the active subpopulation suffers about one break per 5 to 15 kilobases, and about 15 to 20% of the molecules have one or both ends modified. Our analysis of fragmentation is consistent with the random introduction of double-strand breaks, whose cause and exact nature are unknown. Our analysis of end modification indicated that the most prevalent form of damage involved deletion or addition of less than 25 base pairs. In addition we demonstrated directly that the efficiencies of joining sticky, blunt, or mismatched ends are identical, verifying the apparent ability of cells to join nearly any two DNA ends and suggesting that the efficiency of joining approaches 100%. The design of these experiments ensured that the detected damage preceded viral replication and thus should be common to all DNAs transfected with DEAE-dextran and not specific for viral DNA. These measurements of damage within transfected DNA have important consequences for studies of homologous and nonhomologous recombination in somatic cells as is discussed.