Sibani Sahar, Price Gerald B, Zannis-Hadjopoulos Maria
McGill Cancer Center, McGill University, Montreal, Quebec H3G 1Y6, Canada.
J Cell Sci. 2005 Aug 1;118(Pt 15):3247-61. doi: 10.1242/jcs.02427. Epub 2005 Jul 12.
One of the functions of the abundant heterodimeric nuclear protein, Ku (Ku70/Ku80), is its involvement in the initiation of DNA replication through its ability to bind to chromosomal replication origins in a sequence-specific and cell cycle dependent manner. Here, using HCT116 Ku80+/- cells, the effect of Ku80 deficiency on cell cycle progression and origin activation was examined. Western blot analyses revealed a 75% and 36% decrease in the nuclear expression of Ku80 and Ku70, respectively. This was concomitant with a 33% and 40% decrease in chromatin binding of both proteins, respectively. Cell cycle analysis of asynchronous and late G1 synchronized Ku80+/- cells revealed a prolonged G1 phase. Furthermore, these Ku-deficient cells had a 4.5-, 3.4- and 4.3-fold decrease in nascent strand DNA abundance at the lamin B2, beta-globin and c-myc replication origins, respectively. Chromatin immunoprecipitation (ChIP) assays showed that the association of Ku80 with the lamin B2, beta-globin and c-myc origins was decreased by 1.5-, 2.3- and 2.5-fold, respectively, whereas that of Ku70 was similarly decreased (by 2.1-, 1.5- and 1.7-fold, respectively) in Ku80+/- cells. The results indicate that a deficiency of Ku80 resulted in a prolonged G1 phase, as well as decreased Ku binding to and activation of origins of DNA replication.
丰富的异二聚体核蛋白Ku（Ku70/Ku80）的功能之一是通过其以序列特异性和细胞周期依赖性方式结合染色体复制起点的能力参与DNA复制的起始。在此，使用HCT116 Ku80+/-细胞，研究了Ku80缺陷对细胞周期进程和起点激活的影响。蛋白质印迹分析显示，Ku80和Ku70的核表达分别下降了75%和36%。这分别伴随着两种蛋白质与染色质结合的下降33%和40%。对异步和晚期G1期同步的Ku80+/-细胞进行的细胞周期分析显示G1期延长。此外，这些Ku缺陷细胞在核纤层蛋白B2、β-珠蛋白和c-myc复制起点处的新生链DNA丰度分别下降了4.5倍、3.4倍和4.3倍。染色质免疫沉淀（ChIP）分析表明，Ku80与核纤层蛋白B2、β-珠蛋白和c-myc起点的结合分别下降了1.5倍、2.3倍和2.5倍，而在Ku80+/-细胞中Ku70的结合也同样下降（分别下降2.1倍、1.5倍和1.7倍）。结果表明，Ku80缺陷导致G1期延长，以及Ku与DNA复制起点的结合和激活减少。
