Low levels of DNA Ligases III and IV sufficient for effective NHEJ
Low levels of DNA Ligases III and IV sufficient for effective NHEJ
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DOI：
10.1002/jcp.21120
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发表时间：
2007-11-01
影响因子：
5.6
通讯作者：
Iliakis, George
中科院分区：
文献类型：
--
作者：
Windhofer, Frank;Wu, Wenqi;Iliakis, George
高等真核生物的细胞主要通过利用DNA-PKcs、Ku、LIG 4、XRCC 4、XLF/Cernunnos、Artemis以及DNA聚合酶λ（称为D-NHEJ）的产物的非同源DNA末端连接（NHEJ）途径重新连接其DNA中的双链断裂（DSB）。在这些蛋白质中具有缺陷的突变体利用作为备份的NHEJ替代途径（B-NHEJ）从其基因组中去除大部分DSB。虽然D-NHEJ完全依赖于DNA连接酶IV，但最近的工作指出DNA连接酶III是B-NHEJ的组分。在这里，我们使用RNA干扰（RNAi），以进一步调查的NHEJ的途径中的DNA连接酶III和IV的活性要求。我们报道了70-80%的LIG 3表达敲低对DSB重新连接没有可检测的影响，无论是在D-NHEJ熟练的细胞中，还是在D-NHEJ已经被化学或遗传学损害的细胞中。令人惊讶的是，LIG 4敲低对修复熟练的细胞也没有影响，但抑制了具有严重损害其活性的亚型LIG 4突变的放射敏感性细胞系中的DSB重新加入。结果表明，D-NHEJ或B-NHEJ的完全覆盖由非常低的连接酶水平提供，并且证明在具有LIG 4突变的患者的细胞中通过DNA连接酶IV的残余末端连接。
Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non-homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA-PKcs, Ku, LIG4, XRCC4, XLF/Cernunnos, Artemis as well as DNA polymerase lambda (termed D-NHEJ). Mutants with defects in these proteins remove a large proportion of DSBs from their genome utilizing an alternative pathway of NHEJ that operates as a backup (B-NHEJ). While D-NHEJ relies exclusively on DNA ligase IV, recent work points to DNA ligase III as a component of B-NHEJ. Here, we use RNA interference (RNAi) to further investigate the activity requirements for DNA ligase III and IV in the pathways of NHEJ. We report that 70-80% knock down of LIG3 expression has no detectable effect on DSB rejoining, either in D-NHEJ proficient cells, or in cells where D-NHEJ has been chemically orgenetically compromised. Surprisingly, also LIG4 knock down has no effect on repair proficient cells, but inhibits DSB rejoining in a radiosensitive cell line with a hypomorphic LIG4 mutation that severely compromises its activity. The results suggest that complete coverage for D-NHEJ or B-NHEJ is afforded by very low ligase levels and demonstrate residual end joining by DNA ligase IV in cells of patients with mutations in LIG4.