Extrachromosomal Recombination in Mammalian Cells as Studied with Single- and Double-Stranded DNA Substrates

Abstract

We have previously proposed a model to account for the high levels of homologous recombination that can occur during the introduction of DNA into mammalian cells (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984). An essential feature of that model is that linear molecules with ends appropriately located between homologous DNA segments are efficient substrates for an exonuclease that acts in a 5′→3′ direction. That process generates complementary single strands that pair in homologous regions to produce an intermediate that is processed efficiently to a recombinant molecule. An alternative model, in which strand degradation occurs in the 3′→5′ direction, is also possible. In this report, we describe experiments that tested several of the essential features of the model. We first confirmed and extended our previous results with double-stranded DNA substrates containing truncated herpesvirus thymidine kinase (tk) genes (tk Δ 5′ and tk Δ 3′). The results illustrate the importance of the location of double-strand breaks in the successful reconstruction of the tk gene by recombination. We next transformed cells with pairs of single-stranded DNAs containing truncated tk genes which should anneal in cells to generate the recombination intermediates predicted by the two alternative models. One of the intermediates would be the favored substrate in our original 5′→3′ degradative model and the other would be the favored substrate in the alternative 3′→5′ degradative model. Our results indicate that the intermediate favored by the 3′→5′ model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.