G2-phase chromatid break kinetics in irradiated DNA repair mutant hamster cell lines using calyculin-induced PCC and colcemid-block

Abstract

We have investigated the role of the major pathways of DNA double-strand break (DSB) rejoining in the formation and kinetics of disappearance of chromatid breaks following irradiation in the G2 phase of the cell-cycle. We studied the responses of Chinese hamster cell lines xrs5, UV41 and irs1 with mutations in DNA repair genes XRCC5, ERCC4/XPF and XRCC2, involved in the non-homologous end-joining (NHEJ), single-strand annealing (SSA) and homologous recombination (HR) pathways of DSB rejoining respectively. We have used calyculin-induced PCC to study the kinetics of chromatid breaks in xrs5 and UV41 and wild-type CHOK1 cell line. xrs5 showed an elevated frequency of both spontaneous and radiation-induced chromatid breaks. However, the rate of disappearance of chromatid breaks with time was similar in xrs5 to that in its parental CHO cell line. The results with xrs5 firstly confirm our previous findings using the traditional colcemid-block technique, and secondly they demonstrate the independence of chromatid break kinetics of the radiation-induced cell-cycle checkpoint delay. The lack of correspondence between chromatid break kinetics and the deficiency in DSB rejoining in xrs5 argues strongly for an indirect involvement of DSB in the formation of chromatid breaks. The UV41 strain also showed similar chromatid break frequencies and kinetics to CHOK1 suggesting that the SSA pathway is not involved in the rejoining of DSB in the G2 phase of the cell-cycle. We found it not possible to use calyculin-induced PCC in V79-4 and irs1 cell lines. However, using colcemid block we show an elevation in both spontaneous and radiation-induced chromatid break frequency, and a similar rate of disappearance of G2 chromatid breaks with time after irradiation to its wild-type parental V79 line. Thus, deficiencies in two of the major pathways of DSB rejoining (NHEJ and HR) lead to elevated frequencies of chromatid breaks, but do not significantly influence the kinetics of their disappearance with time. We conclude from these data that chromatid breaks do not represent ‘expanded’ DSB but that they are an indirect consequence of the formation of DSB.