The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site
The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site
复制标题
DOI：
10.1093/jb/mvq020
复制
发表时间：
2010-06-01
影响因子：
2.7
通讯作者：
Kurumizaka, Hitoshi
中科院分区：
文献类型：
--
作者：
Saito, Kengo;Kagawa, Wataru;Kurumizaka, Hitoshi
RAD 52是同源重组（HR）的关键因子，在RAD 51依赖和非依赖的HR途径中都起着重要作用。一些研究表明，RAD 52的功能调节与小泛素样修饰物（SUMO）的蛋白质修饰之间存在联系。然而，SUMO调控RAD 52的分子机制尚不清楚。为了开始研究这一机制，我们通过使用已建立的大肠杆菌类小泛素化系统制备RAD 52-SUMO复合物，鉴定了人RAD 52蛋白类小泛素化的可能靶位点。纯化的复合物的酶消化片段的质谱和氨基酸测序显示，位于靠近C末端的RAD 52的推定的核定位信号被sumoylated。RAD 52-SUMO复合物的生物化学研究表明，在确定的网站sumoylation有没有明显的影响，DNA结合，D-环的形成，ssDNA退火和RAD 51结合活性的RAD 52。另一方面，在人细胞中含有在鉴定的类小泛素化位点处的突变的GFP融合的RAD 52蛋白的可视化显示蛋白质的胞质和核分布的明显差异。这些结果表明，SUMO化在RAD 52的核转运中发挥重要作用的可能性。
RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52.