HOMOLOGY-DIRECTED REPAIR IS A MAJOR DOUBLE-STRAND BREAK REPAIR PATHWAY IN MAMMALIAN-CELLS

Mammalian cells leave been presumed to repair potentially lethal chrom osomal double-strand breaks (DSBs) in large part by processes that do not require homology to the break site. This contrasts with Saccharomy ces cerevisiae where the major DSB repair pathway is homologous recomb ination. Recently, it has beers determined that DSBs in genomic DNA in mammalian cells can stimulate homologous recombination as much as 3 o r 4 orders of magnitude, suggesting that homology-directed repair may play an important role in the repair of chromosomal breaks. To determi ne whether mammalian cells use recombinational repair at a significant level, we have analyzed the spectrum of repair events at a defined ch romosomal break by using direct physical analysis of repair products. When an endonuclease-generated DSB is introduced into one of two direc t repeats, homologous repair is found to account for 30-50% of observe d repair events. Both noncrossover and deletional homologous repair pr oducts ape detected, at approximately a 1:3 ratio. These results demon strate the importance of homologous recombination in the repair of DSB s in mammalian cells. In the remaining observed repair events, DSBs ar e repaired by nonhomologous processes. The nonhomologous repair events generally result in small deletions or insertions at the break site, although a small fraction of events result in larger chromosomal rearr angements. Interestingly, in two insertions, GT repeats were integrate d at one of the broken chromosome ends, suggesting that DSB repair can contribute to the spread of microsatellite sequences in mammalian gen omes.