Biochemical analysis of the human ENA/VASP-family proteins, MENA, VASP and EVL, in homologous recombination
Biochemical analysis of the human ENA/VASP-family proteins, MENA, VASP and EVL, in homologous recombination
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DOI：
10.1093/jb/mvr029
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发表时间：
2011-06-01
影响因子：
2.7
通讯作者：
Kurumizakay，Hitoshi
中科院分区：
文献类型：
--
作者：
Takaku，Motoki;Ueno，Hiroyuki;Kurumizakay，Hitoshi
MENA、Vasp和EVL是ENA/Vasp蛋白家族的成员，参与细胞质肌动蛋白的重塑。以前，我们发现EVL直接与RAD51相互作用，RAD51是双链断裂(DSB)同源重组修复中的关键蛋白，并在体外刺激RAD51介导的重组反应。EVL基因敲除的MCF7细胞显示RAD51-病灶的形成明显减少，提示EVL可能通过RAD51介导的同源重组在DSB修复途径中发挥作用。然而，在EVL基因敲除的细胞中，DSB修复缺陷并不明显，这意味着两个EVL类似物，MENA和VASP，可能补充了人类细胞中的EVL功能。因此，在本研究中，我们提纯了人MENA、Vasp和EVL作为重组蛋白，并在体外比较了它们的生化活性。我们发现这三种蛋白质都具有RAD51结合、DNA结合和DNA-退火酶活性。还观察到RAD51介导的同源配对对所有三种蛋白质的刺激作用。此外，表面等离子体共振分析表明，MENA、VASP和EVL之间存在相互作用。这些结果支持ENA/Vasp家族蛋白在同源重组中功能冗余的观点，并且这三种蛋白都可能参与人类的DSB修复途径。
MENA, VASP and EVL are members of the ENA/VASP family of proteins and are involved in cytoplasmic actin remodeling. Previously, we found that EVL directly interacts with RAD51, an essential protein in the homologous recombinational repair of double-strand breaks (DSBs) and stimulates the RAD51-mediated recombination reactionsin vitro. The EVL-knockdown MCF7 cells exhibited a clear reduction in RAD51-foci formation, suggesting that EVL may function in the DSB repair pathway through RAD51-mediated homologous recombination. However, the DSB repair defects were less significant in the EVL-knockdown cells, implying that two EVL paralogues, MENA and VASP, may complement the EVL function in human cells. Therefore, in the present study, we purified human MENA, VASP and EVL as recombinant proteins, and compared their biochemical activitiesin vitro. We found that all three proteins commonly exhibited the RAD51 binding, DNA binding and DNA-annealing activities. Stimulation of the RAD51-mediated homologous pairing was also observed with all three proteins. In addition, surface plasmon resonance analyses revealed that MENA, VASP and EVL mutually interacted. These results support the ideas that the ENA/VASP-family proteins are functionally redundant in homologous recombination, and that all three may be involved in the DSB repair pathway in humans.