Figure S1 - Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes

RAD50 gene-disrupted mutants are lethal to cells. RAD50?/? cells were conditionally created using the tet-repressible promoter (RAD50?/?/tetRAD50), as described for the generation of MRE11-deficient DT40 cells [13]. (A) Schematic representation of a part of the RAD50 locus, the gene disruption constructs and the configuration of the targeted alleles. Solid boxes indicate the position of exons. Only disrupted exons are indicated. Relevant EcoRI sites and the position of the probe used in Southern-blot analysis are indicated. (B) RAD50 gene-targeting was confirmed by Southern-blot analysis. EcoRI-digested genomic DNA from cells with the indicated genotypes of the Rad50 gene was analyzed, using the probe shown in (A). Positions of hybridizing fragments of the WT and targeted loci are indicated. (C) The tetRAD50 transgene expression was inhibited by the addition of doxycycline (Dox). Rad50 protein levels were reduced by at least 100 fold 24 hours after the addition of doxycycline. (D) Growth curves corresponding to the indicated cell cultures. Experiments were done at least three times. +Dox represents continuous exposure to doxycycline. The cells ceased to proliferate four days after the addition of Dox and eventually all died, indicating that RAD50 plays an essential role in the cellular proliferation of any vertebrate cell. (E) Chromosomal breaks accumulated in Rad50 depleted cells. RAD50?/? cells were exposed to Dox for six days. Fifty mitotic cells were analyzed for each chromosomal analysis. (3.1 MB EPS)