CHROMOSOMAL DOUBLE-STRAND BREAK REPAIR IN KU80-DEFICIENT CELLS

The x-ray sensitive hamster cell line xrs-6 is deficient in DNA double -strand break (DSB) repair and exhibits impaired V(D)J recombination. The molecular defect in this line is in the 80-kDa subunit of the Ku a utoantigen, a protein that binds to DNA ends and recruits the DNA-depe ndent protein kinase to DNA. Using an I-SceI endonu clease expression system, chromosomal DSB repair was examined in xrs-6 and parental CHO- K1 cell lines, A DSB in chromosomal DNA increased the yield of recombi nants several thousand-fold above background in both the xrs-6 and CHO -K1 cells, with recombinational repair of DSBs occurring in as many as 1 of 100 cells electroporated with the endonuclease expression vector . Thus, recombinational repair of chromosomal DSBs can occur at substa ntial levels in mammalian cells and it is not grossly affected in our assay by a deficiency of the Ku autoantigen. Rejoining of broken chrom osome ends (end-joining) near the site of the DSB was also examined. I n contrast to recombinational repair, end-joining was found to be seve rely impaired in the xrs-6 cells, Thus, the Ku protein appears to play a critical role in only one of the chromosomal DSB repair pathways.