INTERFERENCE OF DNA-SEQUENCE DIVERGENCE WITH PRECISE RECOMBINATIONAL DNA-REPAIR IN MAMMALIAN-CELLS

Studies done in prokaryotes and eukaryotes have indicated that DNA seq uence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is ba sed on the acquisition by homologous recombination of endogenous LINE- 1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, on e from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Althoug h L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogeno us LINE-1 elements obtained with L1Rn-3 was the same as the one obtain ed with L1Md-A2 which is on average 95% homologous to the LINE-1 eleme nts of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 we re 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1M d-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had bee n introduced into the exogenous LINE-1 sequences had been precisely re paired in 50% of the recombinants obtained with L1Md-A2. None of the L 1Rn-3 recombinants showed precise gap repair. The simplest interpretat ion of these results is that when free DNA ends are involved in homolo gous recombination in mammalian cells, DNA sequence divergence does no t interfere with initiation of recombination, i.e. strand invasion and priming of DNA synthesis, but it rather influences later step(s) such as to change the nature of the outcome products. This could explain s ome aspects of the structure and evolution of repetitive elements.