Structure-specific endonucleases xpf and mus81 play overlapping but essential roles in DNA repair by homologous recombination.
Structure-specific endonucleases xpf and mus81 play overlapping but essential roles in DNA repair by homologous recombination.
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DOI：
10.1158/0008-5472.can-12-3154
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发表时间：
2013-07-15
期刊：
影响因子：
11.2
通讯作者：
Takeda S
中科院分区：
文献类型：
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作者：
Kikuchi K;Narita T;Pham VT;Iijima J;Hirota K;Keka IS;Mohiuddin;Okawa K;Hori T;Fukagawa T;Essers J;Kanaar R;Whitby MC;Sugasawa K;Taniguchi Y;Kitagawa K;Takeda S
DNA双链断裂(DSB)在姐妹染色单体复制过程中频繁发生，当细胞暴露于包括喜树碱在内的化疗药物时，双链断裂显著增加。这种DSB可以通过与完整的姐妹染色单体的同源重组(HR)有效地修复。因此，HR在细胞增殖和对喜树碱的耐受性中起着关键作用。哺乳动物细胞携带多种结构特异性内切酶，如XPF-ERCC1和MUS81-Eme1，其中XPF和MUS81是酶活性所必需的亚基。在这里，我们通过在没有MUS81同源基因的鸡DT40细胞系中有条件地灭活XPF来展示XPF和MUS81之间的功能重叠。虽然缺乏XPF或MUS81的哺乳动物细胞是活的，但在DT40细胞中XPF失活是致命的，导致自发染色体断裂的数量显著增加。同样，在人类HeLa细胞和小鼠胚胎干细胞中，XPF和MUS81的失活都会导致大量自发的染色体断裂。此外，异位表达人MUS81-Eme1或人XPF-ERCC1异源二聚体逆转了XPF缺陷DT40细胞的表型。这些观察结果表明，XPF-ERCC1和MUS81-Eme1在维持基因组DNA方面存在功能重叠。MUS81-Eme1和XPF-ERCC1都有助于HR的完成，以下数据表明，MUS81-Eme1或XPF-ERCC1的表达减少了XPF缺陷DT40细胞中由喜树碱诱导的染色体断裂数量，并且通过删除XRCC3来阻止HR的早期步骤抑制了XPF缺陷DT40细胞的活力。综上所述，XPF和MUS81在完成人力资源方面的职能基本上是重叠的。
DNA double-strand breaks (DSBs) occur frequently during replication in sister chromatids, and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR hence plays pivotal roles in cellular proliferation and cellular tolerance to camptothecin. Mammalian cells carry several structure-specific endonucleases, such as Xpf-Ercc1 and Mus81-Eme1, in which Xpf and Mus81 are the essential subunits for enzymatic activity. Here we show the functional overlap between Xpf and Mus81 by conditionally inactivating Xpf in the chicken DT40 cell line, which has no Mus81 ortholog. Although mammalian cells deficient in either Xpf or Mus81 are viable, Xpf inactivation in DT40 cells was lethal, resulting in a marked increase in the number of spontaneous chromosome breaks. Similarly, inactivation of both Xpf and Mus81 in human HeLa cells and murine embryonic stem cells caused numerous spontaneous chromosome breaks. Furthermore, the phenotype of Xpf-deficient DT40 cells was reversed by ectopic expression of human Mus81-Eme1 or human Xpf-Ercc1 heterodimers. These observations indicate the functional overlap of Xpf-Ercc1 and Mus81-Eme1 in the maintenance of genomic DNA. Both Mus81-Eme1 and Xpf-Ercc1 contribute to the completion of HR as evidenced by the following data that the expression of Mus81-Eme1 or Xpf-Ercc1 diminished the number of camptothecin-induced chromosome breaks in Xpf-deficient DT40 cells, and preventing early steps in HR by deleting XRCC3 suppressed the inviability of Xpf-deficient DT40 cells. In summary, Xpf and Mus81 have a substantially overlapping function in completion of HR.