Split Dose Recovery Studies using Homologous Recombination Deficient Gene Knockout Chicken B Lymphocyte Cells

To understand the role of proteins involved in DSB repair modulating SLD recovery, chicken B lymphoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52^-/- cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C^-/- cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54^-/- cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52^-/-, XRCC2^-/-, XRCC3^-/-RAD51C^-/-and RAD51D^-/-) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52^-/- was reached at about 1-2 hours after the first dose of radiation and then decreased to a minimum thereafter (5h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2^-/-, XRCC3^-/-, RAD51C^-/-and RAD51D^-/- knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D^-/- and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2^-/-, XRCC3^-/-, RAD51C^-/-and RAD51D^-/- and further delayed when compared with that of RAD52^-/- and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in comparison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery.