Bryant Peter E, Mozdarani Hossein, Marr Christie
Bute Medical School, Bute Medical Buildings, University of St Andrews, St Andrews, Fife KY16 9TS, United Kingdom.
Mutat Res. 2008 Nov 17;657(1):8-12. doi: 10.1016/j.mrgentox.2008.08.003. Epub 2008 Aug 12.
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.
我们研究了DNA双链断裂（DSB）重新连接的主要途径在细胞周期G2期照射后染色单体断裂形成及消失动力学中的作用。我们研究了中国仓鼠细胞系xrs5、UV41和irs1的反应，它们分别在参与DSB重新连接的非同源末端连接（NHEJ）、单链退火（SSA）和同源重组（HR）途径的DNA修复基因XRCC5、ERCC4/XPF和XRCC2中存在突变。我们使用花萼海绵诱癌素诱导的早熟染色体凝集（PCC）来研究xrs5和UV41以及野生型CHOK1细胞系中染色单体断裂的动力学。xrs5显示出自发性和辐射诱导的染色单体断裂频率均升高。然而，xrs5中染色单体断裂随时间消失的速率与其亲本CHO细胞系相似。xrs5的结果首先证实了我们之前使用传统秋水仙酰胺阻断技术的发现，其次表明了辐射诱导的细胞周期检查点延迟的染色单体断裂动力学的独立性。xrs5中染色单体断裂动力学与DSB重新连接缺陷之间缺乏对应关系，有力地证明了DSB间接参与染色单体断裂的形成。UV41菌株也显示出与CHOK1相似的染色单体断裂频率和动力学，表明SSA途径不参与细胞周期G2期DSB的重新连接。我们发现无法在V79 - 4和irs1细胞系中使用花萼海绵诱癌素诱导的PCC。然而，使用秋水仙酰胺阻断，我们显示出自发性和辐射诱导的染色单体断裂频率均升高，并且照射后G2染色单体断裂随时间消失的速率与其野生型亲本V79系相似。因此，DSB重新连接的两个主要途径（NHEJ和HR）的缺陷导致染色单体断裂频率升高，但不会显著影响其随时间消失的动力学。我们从这些数据得出结论，染色单体断裂并不代表“扩展的”DSB，而是DSB形成的间接后果。
