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

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.

非同源末端连接（non-homologous DNA end joining, NHEJ）被公认为哺乳动物细胞内双链断裂（double-strand break, DSB）修复的核心途径，其过程依赖于DNA末端结合的Ku70/80异二聚体（DNA end-binding Ku70/80 heterodimer）。为探究Ku在非同源末端连接中的功能，本研究对比了野生型CHO-K1细胞、Ku80缺陷型xrs6细胞以及经Ku80-cDNA互补的xrs6细胞（xrs6-Ku80）的无细胞提取物在体外（in vitro）重连不同类型双链断裂的能力。相较于两类Ku80功能正常的提取物可高效且精准地重连所有类型的DNA末端，xrs6细胞提取物的非同源末端连接效率与精准度均显著下降。该精准度缺失在带有3'突出端（3'-overhang）且毗邻5'突出端（5'-overhang）或平端（blunt end）的非同源末端构型中表现得最为显著。在Ku80功能正常的提取物中，3'突出端的序列大多可通过DNA填充合成（fill-in DNA synthesis）得以保留；而在xrs6细胞提取物中，这些序列会完全丢失，转而形成在断裂点（breakpoints）处带有微同源序列区域（microhomology patches）的小片段缺失。综上，本研究结果与此前针对Ku缺陷型酵母菌株（Ku-deficient yeast strains）的研究结论一致，表明Ku可作为一种对齐因子，不仅能够提升非同源末端连接的效率，还能保障其修复精准度。此外，在缺失Ku的情况下，还存在一种次级非同源末端连接活性，该活性易出错（error-prone），且可能通过碱基配对相互作用（base pairing interactions）得以辅助。