Losada Raquel, Rivero María Teresa, Slijepcevic Predrag, Goyanes Vicente, Fernández José Luis
Sección de Genética y Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo (CHUJC), As Xubias 84, 15006-A Coruña, Spain.
Mutat Res. 2005 Feb 15;570(1):119-28. doi: 10.1016/j.mrfmmm.2004.10.009.
The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was applied to analyze the effect of Wortmannin (WM) in the rejoining kinetics of ionizing radiation-induced DNA double-strand breaks (DSBs) in the whole genome and in the long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cell lines. The results indicate that the ITRS blocks from wild-type Chinese hamster cell lines, CHO9 and V79B, exhibit a slower initial rejoining rate of ionizing radiation-induced DSBs than the genome overall. Neither Rad51C nor the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) activities, involved in homologous recombination (HR) and in non-homologous end-joining (NHEJ) pathways of DSB repair respectively, influenced the rejoining kinetics within ITRS in contrast to DNA sequences in the whole genome. Nevertheless, DSB removal rate within ITRS was decreased in the absence of Ku86 activity, though at a lower affectation level than in the whole genome, thus homogenizing both rejoining kinetics rates. WM treatment slowed down the DSB rejoining kinetics rate in ITRS, this effect being more pronounced in the whole genome, resulting in a similar pattern to that of the Ku86 deficient cells. In fact, no WM effect was detected in the Ku86 deficient Chinese hamster cells, so probably WM does not add further impairment in DSB rejoining than that resulted as a consequence of absence of Ku activity. The same slowing effect was also observed after treatment of Rad51C and DNA-PKcs defective hamster cells by WM, suggesting that: (1) there is no potentiation of the HR when the NHEJ is impaired by WM, either in the whole genome or in the ITRS, and (2) that this impairment may probably involve more targets than DNA-PKcs. These results suggest that there is an intragenomic heterogeneity in DSB repair, as well as in the effect of WM on this process.
采用DNA断裂检测-荧光原位杂交（DBD-FISH）方法，分析渥曼青霉素（WM）对中国仓鼠细胞系全基因组及长间质端粒重复序列（ITRS）区域电离辐射诱导的DNA双链断裂（DSB）重新连接动力学的影响。结果表明，野生型中国仓鼠细胞系CHO9和V79B的ITRS区域，电离辐射诱导的DSB初始重新连接速率比全基因组整体更慢。分别参与DSB修复的同源重组（HR）和非同源末端连接（NHEJ）途径的Rad51C和DNA依赖性蛋白激酶催化亚基（DNA-PKcs）的活性，与全基因组中的DNA序列相比，均不影响ITRS区域内的重新连接动力学。然而，在缺乏Ku86活性时，ITRS区域内的DSB去除率降低，尽管影响程度低于全基因组，从而使两者的重新连接动力学速率趋于一致。WM处理减缓了ITRS区域内DSB的重新连接动力学速率，这种效应在全基因组中更为明显，导致与Ku86缺陷细胞类似的模式。事实上，在Ku86缺陷的中国仓鼠细胞中未检测到WM效应，因此WM可能不会比因缺乏Ku活性而导致的DSB重新连接损伤造成更大的损害。在WM处理Rad51C和DNA-PKcs缺陷的仓鼠细胞后也观察到了相同的减缓效应，这表明：（1）当NHEJ被WM损害时，无论是在全基因组还是在ITRS区域，HR均无增强；（2）这种损害可能涉及比DNA-PKcs更多的靶点。这些结果表明，DSB修复存在基因组内异质性，以及WM对该过程的影响也存在异质性。
