Analysis of the Activities of RAD54, a SWI2/SNF2 Protein, Using a Specific Small-molecule Inhibitor
Analysis of the Activities of RAD54, a SWI2/SNF2 Protein, Using a Specific Small-molecule Inhibitor
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DOI：
10.1074/jbc.m113.502195
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发表时间：
2013-11-01
影响因子：
4.8
通讯作者：
Mazin, Alexander V.
中科院分区：
文献类型：
--
作者：
Deakyne, Julianna S.;Huang, Fei;Mazin, Alexander V.
RAD 54是一种重要的同源重组蛋白，属于ATP酶依赖性DNA移位酶SWI 2/SNF 2家族。在体外，RAD 54刺激RAD 51介导的DNA链交换，并促进霍利迪连接的分支迁移。据认为，ATP酶依赖性DNA易位是这两种RAD 54活性所必需的。在这里，我们确定，通过高通量筛选，一个特定的RAD 54抑制剂，链黑菌素（SN），并使用它来研究RAD 54活动的机制。我们发现SN通过与RAD 54的直接相互作用和活性氧的产生特异性靶向RAD 54 ATP酶，但不靶向DNA结合。与分支迁移（BM）对RAD 54的ATP酶依赖性DNA转位的依赖性一致，SN抑制RAD 54 BM。令人惊讶的是，RAD 54刺激RAD 51 DNA链交换的能力不受SN的显著影响，表明RAD 54 DNA易位在该过程中的作用相对较小。因此，SN的使用使我们能够确定RAD 54 ATP酶和DNA易位对RAD 54的两个主要活性的影响的重要差异，即Holliday连接的BM和DNA配对的刺激。
RAD54, an important homologous recombination protein, is a member of the SWI2/SNF2 family of ATPase-dependent DNA translocases. In vitro, RAD54 stimulates RAD51-mediated DNA strand exchange and promotes branch migration of Holliday junctions. It is thought that an ATPase-dependent DNA translocation is required for both of these RAD54 activities. Here we identified, by high-throughput screening, a specific RAD54 inhibitor, streptonigrin (SN), and used it to investigate the mechanisms of RAD54 activities. We found that SN specifically targets the RAD54 ATPase, but not DNA binding, through direct interaction with RAD54 and generation of reactive oxygen species. Consistent with the dependence of branch migration (BM) on the ATPase-dependent DNA translocation of RAD54, SN inhibited RAD54 BM. Surprisingly, the ability of RAD54 to stimulate RAD51 DNA strand exchange was not significantly affected by SN, indicating a relatively smaller role of RAD54 DNA translocation in this process. Thus, the use of SN enabled us to identify important differences in the effect of the RAD54 ATPase and DNA translocation on two major activities of RAD54, BM of Holliday junctions and stimulation of DNA pairing.