Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex
Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex
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DOI：
10.1266/ggs.89.169
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发表时间：
2014-08-01
影响因子：
1.1
通讯作者：
Kurumizaka, Hitoshi
中科院分区：
文献类型：
--
作者：
Kobayashi, Wataru;Sekine, Satoshi;Kurumizaka, Hitoshi
绿色荧光蛋白 (GFP) 融合到目标蛋白质的 N 或 C 末端，广泛用于监测细胞中蛋白质的定位和流动性。 RAD51 是一种必需蛋白，通过促进同源重组反应在有丝分裂 DNA 修复和减数分裂染色体分离中发挥作用。先前对拟南芥的遗传学研究表明，融合到 RAD51 C 末端的 GFP (RAD51-GFP) 会抑制有丝分裂 DNA 修复，但减数分裂同源重组不受影响。为了确定 C 端 GFP 如何特异性抑制 RAD51 有丝分裂 DNA 修复，我们纯化了水稻 RAD51A1-GFP 和 RAD51A2-GFP，并进行了生化分析。有趣的是，纯化的 RAD51A1-GFP 和 RAD51A2-GFP 擅长 DNA 结合和 ATP 水解。然而，含有单链DNA和RAD51A1-GFP或RAD51A2-GFP的核蛋白复合物在与第二个DNA分子的结合（次级DNA结合）方面存在显着缺陷，因此无法催化同源配对。相比之下，RAD51A1-GFP 和 RAD51A2-GFP 有效地刺激了由减数分裂特异性 RAD51 同工型 DMC1 促进的同源配对。这些生化特征在人类 RAD51-GFP 中得到很好的保存。因此，融合到 RAD51 C 末端的 GFP 通过破坏二级 DNA 结合而消除了 RAD51 的同源配对活性，但不影响其 DMC1 刺激活性。
Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses. Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.