Duan Zhiqiang, Zhao Jiafu, Xu Houqiang, Xu Haixu, Ji Xinqin, Chen Xiang, Xiong Jianming
Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China; College of Animal Science, Guizhou University, Guiyang 550025, China.
Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China; College of Animal Science, Guizhou University, Guiyang 550025, China.
Arch Biochem Biophys. 2017 Nov 15;634:57-68. doi: 10.1016/j.abb.2017.09.019. Epub 2017 Oct 7.
Numerous studies have shown that nuclear localization of BLM protein, a member of the RecQ helicases, mediated by nuclear localization signal (NLS) is critical for DNA recombination, replication and transcription, but the mechanism by which BLM protein is imported into the nucleus remains unknown. In this study, the nuclear import pathway for BLM was investigated. We found that nuclear import of BLM was inhibited by two dominant-negative mutants of importin β1 and NTF2/E42K, which lacks the ability to bind Ran and RanGDP, respectively, but was not inhibited by the Ran/Q69L, which is deficient in GTP hydrolysis. Further studies revealed that nuclear import of BLM was reconstituted using importin β1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. Moreover, BLM had direct binding to importin β1 through its NLS domain with the 14-16 HEAT repeats of importin β1. Furthermore, importin β1, Ran or NTF2 depletion by siRNA disrupted the accumulation of BLM protein in the nucleus. These results showed that BLM enters the nucleus via the importin β1, RanGDP and NTF2 dependent pathway, demonstrating for the first time the nuclear trafficking mechanism of a DNA helicase.
众多研究表明，RecQ解旋酶家族成员BLM蛋白经核定位信号（NLS）介导的核定位对于DNA重组、复制及转录至关重要，但BLM蛋白导入细胞核的机制仍不清楚。在本研究中，对BLM的核输入途径进行了研究。我们发现，importin β1和NTF2/E42K的两个显性负性突变体分别抑制了BLM的核输入，这两个突变体分别缺乏结合Ran和RanGDP的能力，但缺乏GTP水解能力的Ran/Q69L并未抑制BLM的核输入。进一步研究表明，在洋地黄皂苷通透的HeLa细胞中，使用importin β1、RanGDP和NTF2可重建BLM的核输入。此外，BLM通过其NLS结构域与具有14 - 16个HEAT重复序列的importin β1直接结合。此外，通过siRNA使importin β1、Ran或NTF2缺失会破坏BLM蛋白在细胞核中的积累。这些结果表明，BLM通过依赖importin β1、RanGDP和NTF2的途径进入细胞核，首次证明了一种DNA解旋酶的核运输机制。
