DNA double-strand break rejoining in xrs5 cells is more rapid in the G2 than in the G1 phase of the cell cycle

Abstract

The radiosensitive xrs5 mutant cell line of CHO K1 shows an overall deficiency in DNA double-strand break (dsb) rejoining. However, xrs5 paradoxically shows an apparently normal rate of disappearance of chromatid breaks with time, the kinetics of which is thought to reflect the underlying rejoining of dsb. Nevertheless the yield of chromatid breaks is elevated by four-fold in xrs5. A possible explanation of the paradox might be that xrs5 is proficient in rejoining dsb in the G2 phase of the cell cycle but converts a higher number of dsb into chromatid breaks. In order to test this we have measured the rejoining of dsb in partially synchronised G2 xrs5 cells and compared the kinetics with those of cells synchronised in the G1 phase. Synchronisation of cells was achieved in G2 by release of cells from an aphidicolin block, and in G1 by staurosporine block. Cell synchrony was monitored by cytofluorometry and showed typically a 67% synchronization of G2 cells and a 91% synchronisation of G1 cells. Rejoining of dsb was measured using neutral filter elution at pH 9.6. G2 cells showed a two-component kinetic with t1/2 values of 9 min and 3.6 h for dsb rejoining. Corresponding t1/2 values for G1 cells were 15 min and approximately 8.8 h. The t1/2 value of 3.6 h found for dsb rejoining in G2 cells is similar to a previously published value for asynchronous parental CHO K1 cells of approximately 4 h. The kinetics of chromatid break rejoining was measured in both xrs5 and CHO K1 following a dose of 0.75 Gy. The kinetics were found to be similar (t1/2 = 2.4 h) in the two cell lines, as previously reported using an equiclastogenic dose.