Wang Huichen, Perrault Ange Ronel, Takeda Yoshihiko, Qin Wei, Wang Hongyan, Iliakis George
Department of Radiation Oncology, Division of Experimental Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107, USA.
Nucleic Acids Res. 2003 Sep 15;31(18):5377-88. doi: 10.1093/nar/gkg728.
Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a non-homologous end joining (NHEJ) apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4 and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with an order of magnitude slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group and frequently joins incorrect ends. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. The present study investigates the role of Ku in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient, error-free, end joining observed in such in vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite the fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA end joining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing end joining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts in line with the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the affinity of Ku for DNA ends, particularly when cooperating with DNA-PKcs, suppresses B-NHEJ by quickly and efficiently binding DNA ends and directing them to D-NHEJ for rapid joining. A chromatin-based model of DNA DSB rejoining accommodating biochemical and genetic results is presented and deviations between in vitro and in vivo results discussed.
高等真核生物的细胞在数分钟内利用一种非同源末端连接（NHEJ）机制处理其基因组中的双链断裂（DSB），该机制涉及DNA-PKcs、Ku、DNA连接酶IV、XRCC4以及其他尚未明确的因子。尽管这些因子中的任何一个受到化学抑制或发生突变都会延迟处理过程，但细胞最终会利用另一条动力学速度慢一个数量级的替代途径去除大部分DNA双链断裂。这条替代途径在RAD52上位性组基因缺陷的突变体中具有活性，并且经常连接错误的末端。因此，我们提出，它反映了一种替代形式的NHEJ，作为DNA-PK依赖（D-NHEJ）途径的备份（B-NHEJ）发挥作用，而不是DSB的同源定向修复。本研究以全细胞提取物中限制性内切酶线性化质粒DNA的末端连接为模型，研究了Ku在这些途径协调中的作用。在此类体外反应中观察到的高效、无错误的末端连接受到抗Ku抗体的强烈抑制。尽管在存在抗体或不存在DNA-PKcs的情况下，Ku都能有效结合DNA末端，但这种抑制作用仍需要DNA-PKcs。wortmannin（一种DNA-PKcs抑制剂）在存在Ku但不存在Ku时也能介导对DNA末端连接的强烈抑制，并且这种抑制作用可以通过用双链寡核苷酸预孵育反应来挽救。这些结果与Ku在通过有效末端结合和与DNA-PKcs的有效相互作用介导末端连接到DNA-PK依赖途径中的作用相一致。另一方面，在缺乏DNA-PKcs的细胞提取物以及Ku耗尽的提取物中观察到了高效的末端连接，这与替代途径的运作一致。耗尽Ku和DNA-PKcs的提取物以相似的效率重新连接平端以及具有3'或5'突出单链的同源末端，但添加Ku会抑制平端和具有3'突出端的同源末端的连接。我们提出，Ku对DNA末端的亲和力，特别是在与DNA-PKcs协同作用时，通过快速有效地结合DNA末端并将其引导至D-NHEJ进行快速连接，从而抑制B-NHEJ。本文提出了一个基于染色质的DNA DSB重新连接模型，该模型兼顾了生化和遗传结果，并讨论了体外和体内结果之间的差异。
