BLAP75蛋白与DNA螺旋酶BLM以及拓扑异构酶TopoⅢα的相互作用

摘要

Objective To identify the interaction site of BLAP75 with BLM or Topo Ⅲα. Methods A series of BLAP75 expression vectors with MBP tag were constructed . The interaction site of BLAP75 , BLM and Topo Ⅲ α was determined by in vitro protein-protein binding assay and confirmed by in vivo immunoprecipitation. Western blotting was used to detect the protein levels of BLM and Topo Ⅲ α after the expression of BLAP75 was specifically suppressed by siRNA. Results BLM and Topo Ⅲ α could bind to BLAP75 through its amino -terminal domain. The interaction between BLAP75 and BLM was mediated by DNA. The protein levels of BLM and Topo Ⅲ α were both decreased when endogenous BLAP 75 was knocked down. Conclusion The N-terminal region of BLAP75 mediates the interaction with BLM and Topo Ⅲ α in cells and BLAP75 helps to maintain the protein levels of BLM and Topo Ⅲ α.%目的 确定BLAP75蛋白与BLM蛋白及TopoⅢα蛋白的相互作用及作用位点.方法 构建一系列携带MBP标记的不同BLAP75载体,通过体外纯化蛋白结合实验检测BLAP75与BLM、TopoⅢα的相互作用位点,并在真核细胞中通过免疫共沉淀方法进行验证;运用RNA干扰(RNAi)技术特异性抑制BLAP75的表达,并利用Western印迹法检测其对BLM和TopoⅢα蛋白表达的影响.结果 BLAP75与BLM和TopoⅢα蛋白的结合位点位于BLAP75的N端区域; BLAP75与BLM的直接相互作用需要DNA的介导;当BLAP75蛋白表达量减少时,BLM和TopoⅢα蛋白量也随之减少.结论 细胞内BLM和TopoⅢα蛋白均结合在BLAP75的氨基端部分;BLAP75对TopoⅢα和BLM的蛋白水平具有重要影响.