DIFFERENT CAPACITIES FOR RECOMBINATION IN CLOSELY-RELATED HUMAN LYMPHOBLASTOID CELL-LINES WITH DIFFERENT MUTATIONAL RESPONSES TO X-IRRADIATION

WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resista nt to the cytotoxic effects of S-irradiation but considerably more sen sitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of th ymidine kinase (tk)-deficient mutants isolated after X-ray treatment o f tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Sout hern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity. (LOH), all 106 mutants from WIL2-NS derivat ives arose with LOB at tk and all but one showed LOH at other linked l oci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one fk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were ana lyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lin es arose by deletion. The remaining mutants retained two copies of the rk gene and thus arose by a mechanism involving the homologous allele . Since many of these mutants arising by a homologous mechanism retain ed partial heterozygosity of chromosome 17, they must have arisen by r ecombination or gene conversion, and not chromosome loss and reduplica tion. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were gl eater in WIL2-NS tha n in TK6. These data are consistent with a model suggesting that a rec ombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS result s from more frequent inter- and intramolecular recombination events.