DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining
作者:E. Feldmann、V. Schmiemann、W. Goedecke、S. Reichenberger、P. Pfeiffer
DOI:10.1093/nar/28.13.2585
日期:2000.7.1
Non-homologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 heterodimer. To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3′-overhangs that abut a 5′-overhang or blunt end. While the sequences of the 3′-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. In summary, our results are consistent with previous results from Ku-deficient yeast strains and indicate that Ku may serve as an alignment factor that not only increases NHEJ efficiency but also accuracy. Furthermore, a secondary NHEJ activity is present in the absence of Ku which is error-prone and possibly assisted by base pairing interactions.
非同源 DNA 末端连接 (NHEJ) 被认为是哺乳动物细胞中双链断裂 (DSB) 修复的主要途径,并且取决于 DNA 末端结合 Ku70/80 异二聚体等。为了研究 Ku 在 NHEJ 中的功能,我们比较了野生型 CHO-K1 细胞、Ku80 缺陷型 xrs6 细胞和 Ku80-cDNA 互补的 xrs6 细胞 (xrs6-Ku80) 的无细胞提取物重新加入不同类型的细胞的能力。 DSB 体外。虽然两种 Ku80 丰富的提取物在重新连接所有类型的 DNA 末端时非常高效且准确,但 xrs6 提取物却显示出 NHEJ 效率和准确度大幅下降。在包含与 5' 突出端或平端相连的 3' 突出端的非同源末端构型中,准确性的缺乏最为明显。虽然 3'-突出端的序列大部分通过填充 DNA 合成在 Ku80 熟练的提取物中得以保留,但它们在 xrs6 提取物中总是完全丢失,因此,相反,在其断点处出现显示微同源补丁的小缺失。总之,我们的结果与 Ku 缺陷酵母菌株的先前结果一致,表明 Ku 可能作为比对因子,不仅提高 NHEJ 效率,而且提高准确性。此外,在缺乏 Ku 的情况下存在二级 NHEJ 活性,这很容易出错,并且可能通过碱基配对相互作用来辅助。