Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding
Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding
复制标题
DOI：
10.1074/jbc.m607180200
复制
发表时间：
2007-03-09
影响因子：
4.8
通讯作者：
Hirose, Fumiko
中科院分区：
文献类型：
--
作者：
Yamashita, Daisuke;Komori, Hirofumi;Hirose, Fumiko
我们之前证明，hDREF 是果蝇 DNA 复制相关元件结合因子 (dDREF) 的人类同源物，是一种主要分布在细胞核中颗粒结构的 DNA 结合蛋白。在这里，谷胱甘肽 S-转移酶下拉和化学交联测定表明，hDREF 的羧基末端 hATC 结构域在 hAT 转座酶家族成员中高度保守，具有自缔合活性。免疫沉淀分析表明，hDREF 在体内自关联，依赖于 hATC 结构域。此外，使用一系列在 hATC 结构域中进行氨基酸取代的 hDREF 突变体进行的分析表明，保守的疏水性氨基酸对于自缔合至关重要。免疫荧光研究进一步表明，所有缺乏自联活性的hDREF突变体均未能在细胞核中积累。自缔合缺陷的 hDREF 突变体也失去了与内源性输入蛋白 β1 的关联。此外，电泳凝胶迁移率变化分析表明，这些突变完全消除了 hDREF 的 DNA 结合活性。这些结果表明 hDREF 通过 hATC 结构域的自缔合对于其核积累和 DNA 结合是必要的。我们还发现人类 hAT 家族的另一个成员 ZBED4/KIAA0637 也可以自关联，同样依赖于 hATC 结构域，缺失会导致有效核积累的损失。因此，人类 hAT 家族成员的 hATC 结构域似乎在核积累所需的自缔合中具有保守功能。
We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta 1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.