Modification of DNA ends can decrease end joining relative to homologous recombination in mammalian cells.
In animal cells, exogenous DNA recombines into random chromosomal sites much more frequently than it recombines into homologous sites. Free DNA ends are "recombinogenic" in both processes. To test the effects of specific ends on analogous extrachromosomal processes, we constructed a linear genome of simian virus 40 with terminal repeated sequences. After transfection into monkey cells, the model substrate can circularize by end joining (analogous to random integration) or by homologous recombination between its terminal repeats (analogous to targeted recombination). Since the two types of recombination are in competition with one another, the ratio of homologous-recombination to end-join products is a sensitive indicator of the differential effects of specific ends. Substrates with blunt ends, complementary sticky ends, or mismatched ends generated the same ratio of homologous-recombination to end-join products. However, addition of dideoxynucleotides to the 3' hydroxyls of the substrate decreased the frequency of end joining by a factor of 5-6 relative to homologous recombination. Thus, the frequency of end joining can be decreased relative to that of homologous recombination by modification of the ends of the input DNA. These results suggest an approach to altering the ratio of random to targeted integration in mammalian cells.
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004270:DNA, Circular / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral