NONHOMOLOGOUS RECOMBINATION IN HUMAN-CELLS
NONHOMOLOGOUS RECOMBINATION IN HUMAN-CELLS
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DOI：
10.1128/mcb.14.1.156
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发表时间：
1994-01-01
影响因子：
5.3
通讯作者：
FISHEL, R
中科院分区：
文献类型：
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作者：
DERBYSHIRE, MK;EPSTEIN, LH;FISHEL, R
非同源重组(NHR)是体细胞DNA染色体双链断裂修复的主要途径。本研究比较了体内转染腺病毒DNA片段的非同源末端连接与纯化的人蛋白在体外催化非同源末端连接的能力。腺病毒DNA片段被证明在人类细胞中有效地连接，而不管末端的结构如何。对这些连接的序列分析表明，两个参与末端经常丢失连接部位3‘链上的核苷酸。为了检查末端连接的生化基础，从各种哺乳动物细胞系中制备了核提取液，并测试了它们连接测试质粒底物的能力。观察到线性底物DNA的有效连接，体外产物在连接处3‘核苷酸的丢失方面与体内产物相似。用人未成熟T细胞系HPB-ALL进行了末端连接活性的大量纯化。蛋白质制剂被发现连接了所有类型的含有异源末端的线性DNA底物，效率接近相等。根据一些标准，体外末端连接系统似乎不包含已知的三种DNA连接酶中的任何一种，并被称为NHR连接酶。丰富的活性存在于一个高相对分子质量的重组复合体中，它似乎包括并需要人类同源配对蛋白HPP-1以及NHR连接酶。对NHR连接酶反应产物分子的表征表明，它们是非随机连接的单体底物的线形低聚物。连接产物的连接端在连接前被3‘外切酶修饰，没有检测到环状DNA分子。这些类型的产物类似于断裂-融合-桥周期所需的产物，断裂-融合-桥周期是染色体双链断裂修复的主要NHR途径。
Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.