Linear DNA must have free ends to transform rat cells efficiently.
We have observed that failure to remove certain restriction enzymes after digestion reduced the transforming ability of DNA from 10- to 50-fold. The DNA found integrated in the transformed cells isolated under these conditions had lost little or no sequences. We interpret these results as indicating that certain restriction enzymes remain bound to the DNA ends after digestion, thus generating a substrate unfavorable both for integration and exonucleolytic degradation. As expected from this interpretation, removal of the restriction enzymes before transfection restored the full transforming ability of linear DNA, but also resulted in the integrated sequences being significantly shorter than the transfected DNA. These findings strongly argue for the hypothesis that integration of linear DNA by illegitimate recombination requires free ends and further suggest that exonucleolytic degradation of such ends may generate a preferred substrate for integration. Finally, a comparison of the sequences found integrated after transfection with circular or linear molecules, led us to conclude that circular molecules need not be linearized to become integrated.
D000818:Animals; D001483:Base Sequence; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics*; D005814:Genes, Viral; D011120:Polyomavirus / Q000235:genetics*; D014162:Transfection
D004279:DNA, Viral; D004262:DNA Restriction Enzymes