ILLEGITIMATE RECOMBINATION INDUCED BY DNA DOUBLE-STRAND BREAKS IN A MAMMALIAN CHROMOSOME

We examined DNA double-strand-break-induced mutations in the endogenou s adenine phosphoribosyltransferase (APRT) gene in cultured Chinese ha mster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand brea ks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT lo cus. Colonies of viable cells containing mutations at APRT were expand ed, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consis ted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small dele tions involved overlaps of one to four complementary bases at the reco mbination junctions. Southern blot analysis revealed more complex muta tions, suggesting translocation, inversion, or insertion of larger chr omosomal fragments. These results indicate that blunt-end DNA double-s trand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagene sis.