Division of Biochemistry, Graduate School of Pharmaceutical Sciences, Setsunan University, Osaka 573–0101, Japan.
Biol Pharm Bull. 2013;36(7):1159-66. doi: 10.1248/bpb.b13-00136.
RecQ5, a member of the RecQ helicase family, maintains genome stability via participation in many DNA metabolic processes including DNA repair, DNA resolution, and RNA transcription, processes occurring in the nucleus. Previously, we reported that RecQ5 and Rad51, also involved in DNA repair, become co-localized in nuclei when co-expressed in cultured cells. Nuclear localization of RecQ5 appears to be important for cellular function along with Rad51. However, little is known about the nuclear localization of RecQ5. Here, we generated enhanced green fluorescent protein (EGFP)-tagged RecQ5 transgenic flies and analyzed localization of this protein in early embryos by live imaging. In syncytial embryos, RecQ5 was localized synchronously in interphase nuclei, and spread repeatedly over the embryos in mitosis. Thus, RecQ5 was transported into nuclei at the early interphase. Furthermore, we examined the subcellular localization of a series of truncated forms of Drosophila RecQ5 in cultured cells to determine the nuclear localization signal (NLS). Entire coding or deleted RecQ5 sequences of various sizes were ligated into EGFP vectors, which were then used to transfect cultured Drosophila cells. The region responsible for nuclear localization of Drosophila RecQ5 contained a short stretch of positively charged basic amino acids, 2 of which were particularly important for the nuclear localization. This stretch was sufficient for nuclear localization when fused with EGFP. Although the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence, this fly RecQ5 protein was translocated into the nucleus by an NLS.
RecQ5 是 RecQ 解旋酶家族的成员，通过参与包括 DNA 修复、DNA 解析和 RNA 转录在内的许多 DNA 代谢过程来维持基因组稳定性，这些过程发生在细胞核中。此前，我们报道称，RecQ5 和 Rad51 也参与 DNA 修复，当在培养细胞中共表达时，它们会在核内共定位。RecQ5 的核定位对于细胞功能与 Rad51 一样重要。然而，人们对 RecQ5 的核定位知之甚少。在这里，我们生成了增强型绿色荧光蛋白 (EGFP) 标记的 RecQ5 转基因果蝇，并通过活细胞成像分析该蛋白在早期胚胎中的定位。在合胞体胚胎中，RecQ5 在间期核中同步定位，并在有丝分裂中反复扩散到胚胎中。因此，RecQ5 在早期间期被转运到核内。此外，我们在培养的细胞中检查了一系列果蝇 RecQ5 的截断形式的亚细胞定位，以确定核定位信号 (NLS)。各种大小的完整编码或缺失 RecQ5 序列被连接到 EGFP 载体中，然后用于转染培养的果蝇细胞。果蝇 RecQ5 的核定位区域包含一段短的带正电荷的碱性氨基酸，其中 2 个氨基酸对核定位特别重要。当与 EGFP 融合时，该片段足以进行核定位。尽管果蝇 RecQ5 的 NLS 在位置和氨基酸序列上与人类 RECQL5 的 NLS 不同，但这种果蝇 RecQ5 蛋白通过 NLS 被转运到核内。
