MODEL FOR HOMOLOGOUS RECOMBINATION DURING TRANSFER OF DNA INTO MOUSE L-CELLS - ROLE FOR DNA ENDS IN THE RECOMBINATION PROCESS
MODEL FOR HOMOLOGOUS RECOMBINATION DURING TRANSFER OF DNA INTO MOUSE L-CELLS - ROLE FOR DNA ENDS IN THE RECOMBINATION PROCESS
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DOI：
10.1128/mcb.4.6.1020
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发表时间：
1984-01-01
影响因子：
5.3
通讯作者：
STERNBERG, N
中科院分区：
文献类型：
--
作者：
LIN, FL;SPERLE, K;STERNBERG, N
噬菌体.lambda。质粒DNA底物(.lambda。tk2和ptk2)含有2个缺陷的疱疹病毒胸苷激酶（tk）基因，可用于检测DNA转移到胸苷激酶活性缺乏的小鼠L细胞中的同源重组。重组事件重建了一个野生型tk基因，并被评分，因为它将tk -细胞转化为tk +。利用该系统可以检测基因转移后的分子内和分子间同源重组；重组程度随tk基因同源性的降低而降低；利用限制性内切酶在相对于重组序列的适当位点切割tk2 DNA，可使重组效率提高10- 100倍。基于这些重组事件的底物要求，提出了一个模型来解释重组如何发生在哺乳动物DNA链降解的外切酶中。这个过程暴露了两个有缺陷的tk基因的互补链，然后它们配对。在配对和未配对区域之间的连接处去除未配对的DNA允许间隙修复过程来重建完整的基因。
Phage .lambda. and plasmid DNA substrates (.lambda.tk2 and ptk2) were constructed that contain 2 defective herpes virus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity. The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+. Using this system, both intramolecular and intermolecular homologous recombination can be detected after gene transfer; the degree of recombination decreases with decreasing tk gene homology; and the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction nezymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, a model is proposed to explain how recombination might occur im mammalian exonucleases that degrade DNA strands. This process exposes complementary strands of the 2 defective tk genes, which then pair. Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.