Biochemical evidence for Ku-independent backup pathways of NHEJ (Publication with Expression of Concern)
Biochemical evidence for Ku-independent backup pathways of NHEJ (Publication with Expression of Concern)
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DOI：
10.1093/nar/gkg728
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发表时间：
2003-09-15
影响因子：
14.9
通讯作者：
Iliakis, G
中科院分区：
文献类型：
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作者：
Wang, HC;Perrault, AR;Iliakis, G
高等真核生物的细胞利用非同源末端连接（NHEJ）装置在几分钟内处理其基因组中的双链断裂（DSBs），该装置涉及DNA- pkcs， Ku， DNA连接酶IV， XRCC4和其他尚未确定的因素。虽然化学抑制或突变，在这些因素中的任何一个延迟处理，细胞最终去除大部分DNA dsb使用一个替代途径，以一个数量级慢的动力学操作。这一替代途径在RAD52上位性组基因缺乏的突变体中是活跃的，并且经常连接错误的末端。因此，我们提出，它反映了NHEJ的另一种形式，作为dna - pk依赖性（D-NHEJ）途径的备份（B-NHEJ），而不是同源性定向修复dsb。本研究利用全细胞提取物中限制性内切酶线性化质粒DNA的末端连接模型，研究了Ku在这些途径协调中的作用。在这种体外反应中观察到的高效，无错误，末端连接被抗ku抗体强烈抑制。这种抑制需要DNA- pkcs，尽管Ku在抗体存在或DNA- pkcs不存在的情况下有效地结合DNA末端。DNA末端连接的强烈抑制也可由wortmannin介导，wortmannin是DNA- pkcs的一种抑制剂，在Ku存在的情况下而不是在Ku不存在的情况下，这种抑制可以通过双链寡核苷酸预孵育反应来恢复。这些结果与Ku在指导末端连接到DNA-PK依赖途径中的作用是一致的，该途径是通过有效的末端结合和与DNA-PKcs的有效相互作用介导的。另一方面，在缺乏DNA-PKcs的细胞提取物中，以及在贫钾提取物中，观察到有效的末端连接，这与替代途径的运作一致。缺失Ku和DNA-PKcs的提取物能以相似的效率重新连接钝端和3‘或5’伸出单链的同源端，但添加Ku抑制钝端和3'伸出的同源端的连接。我们提出Ku对DNA末端的亲和力，特别是当与DNA- pkcs合作时，通过快速有效地结合DNA末端并引导它们快速连接到D-NHEJ来抑制B-NHEJ。提出了一种基于染色质的DNA DSB重新连接模型，以适应生化和遗传结果，并讨论了体外和体内结果之间的差异。
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.