Open AccessModel for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process.
Author(s) -
F L Lin,
Karen Sperle,
Nat Sternberg
Publication year - 1984
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.4.6.1020
Subject(s) - homologous recombination , biology , flp frt recombination , site specific recombination , non homologous end joining , recombination , dna , cre lox recombination , microbiology and biotechnology , mitotic crossover , in vitro recombination , genetic recombination , thymidine kinase , gene , gene conversion , plasmid , non allelic homologous recombination , restriction enzyme , dna repair , genetics , complementary dna , recombinase , molecular cloning , transgene , virus , genetically modified mouse , herpes simplex virus
We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus 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, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells. The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands. This process exposes complementary strands of the two 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.