In vitro rejoining of DNA double strand breaks:: a comparison of genomic-DNA with plasmid-DNA-based assays
In vitro rejoining of DNA double strand breaks:: a comparison of genomic-DNA with plasmid-DNA-based assays
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DOI：
10.1080/095530098142022
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发表时间：
1998-05-01
影响因子：
2.6
通讯作者：
Iliakis, G
中科院分区：
文献类型：
--
作者：
Cheong, N;Perrault, AR;Iliakis, G
目的：评价以基因组DNA为基础的体外DNA双链断裂(DSB)检测方法与以质粒DNA为基础的体外检测方法所需的酶活性之间的相似性。由于后一种检测方法更简单、更快速，如果两种检测方法都能检测到相同的活性，则应首选该方法来表征修复因子。然而，如果两种方法对DSB重联的酶要求不同，两种方法都应该使用，因为它们在修复因子的表征中可能起到互补的作用。材料和方法：建立了一种无细胞分析方法，用于研究辐射诱导的DNA DSB重连接，该DNA是由琼脂糖包埋细胞制备的，使用HeLa细胞的提取液作为酶的来源。结果：在所用条件下，线性化的质粒DNA的连接和裸露的基因组DNA中的DSB的重新连接涉及不同的活性。DSB在基因组DNA中诱导的最佳连接在37℃下用细胞质细胞提取液观察到，而在25℃下用核提取液观察到最佳连接。DSB在基因组DNA中的连接在14℃时几乎停止，但在这个温度下，质粒DNA连接以显著的速度进行。此外，在基因组DNA中诱导的DSB重新连接所需的活性在50℃加热1h时部分稳定，而连接质粒DNA所需的活性则通过类似的处理完全失活。这两个反应都需要ATP才能获得最佳性能，而且在两个反应中，DNA连接在高浓度时都受到抑制。结论：这些观察表明，基于质粒和基因组DNA的检测至少部分地探测了不同的活性集合，因此有望在DSB重新连接所涉及的活性的纯化和表征中发挥互补作用。
Purpose: To evaluate potential similarities between the enzymatic activities required to rejoin DNA double strand breaks (dsb) in an in vitro assay based on genomic DNA and an in vitro assay based on plasmid DNA. Because the latter assay is simpler and faster, it should be preferred for the characterization of repair factors if both assays are found to probe for the same activities. If, however, the enzymatic requirements for dsb rejoining are different between the two assays, both should be used as they are likely to play complementary roles in the characterization of repair factors.Materials and methods: A cell-free assay has been used, developed to study rejoining DNA dsb induced by radiation in 'naked' DNA prepared from agarose embedded cells using an extract of HeLa cells as a source of enzymes. Also employed was an in vitro assay using the ligation of linearized plasmid DNA to model dsb rejoining.Results: Evidence is presented that, under the conditions employed, different sets of activities are involved in the ligation of linearized plasmid DNA and in the rejoining of dsb in 'naked' genomic DNA. Optimal rejoining of dsb induced in genomic DNA is observed with cytoplasmic cell extract at 37 degrees C, whereas optimal ligation of plasmid DNA is observed with nuclear extract at 25 degrees C. Rejoining of dsb in genomic DNA comes to a near halt at 14 degrees C, but plasmid DNA ligation proceeds at significant rates at this temperature. Furthermore, the activities required for the rejoining of dsb induced in genomic DNA are partly stable to heating at 50 degrees C for 1 h, whereas activities required for the ligation of plasmid DNA are completely inactivated by a similar treatment. Both reactions require ATP for optimal performance, and in both, DNA joining is inhibited at high ATP concentrations.Conclusions: These observations indicate that the plasmid-and the genomic-DNA-based assays probe for, at least partly, different sets of activities and therefore are expected to play complementary roles in the purification and characterization of activities involved in dsb rejoining.