Derbyshire M K, Epstein L H, Young C S, Munz P L, Fishel R
Department of Microbiology and Molecular Genetics, University of Vermont School of Medicine, Burlington 05405.
Mol Cell Biol. 1994 Jan;14(1):156-69. doi: 10.1128/mcb.14.1.156-169.1994.
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.
非同源重组（NHR）是体细胞DNA中染色体双链断裂修复的主要途径。在本研究中，对体内转染的腺病毒DNA片段的非同源末端连接与纯化的人类蛋白质在体外催化非同源末端连接的能力进行了比较。结果表明，无论末端结构如何，腺病毒DNA片段在人类细胞中都能有效地连接。对这些连接点的序列分析表明，参与连接的两个末端在连接部位的3'链上经常丢失核苷酸。为了研究末端连接的生化基础，从多种哺乳动物细胞系中制备了核提取物，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，体外产物在连接部位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对末端连接活性进行了大量纯化。发现该蛋白质制剂能以几乎相同的效率连接所有类型含有异源末端的线性DNA底物。基于多项标准，末端连接的体外系统似乎不包含三种已知的DNA连接酶中的任何一种，因此被称为NHR连接酶。富集的活性存在于一种高分子量重组复合物中，该复合物似乎包含并需要人类同源配对蛋白HPP-1以及NHR连接酶。NHR连接酶反应产物分子的特征表明，它们是单体底物的线性寡聚物，以非随机的头对头和/或尾对尾方式连接。发现产物的连接末端在连接之前被3'核酸外切酶修饰，并且未检测到环状DNA分子。这些类型的产物与断裂-融合-桥循环所需的产物相似，断裂-融合-桥循环是染色体双链断裂修复的主要NHR途径。
