脆性组氨酸三联体与复制蛋白A相互作用关键氨基酸残基的鉴定  

Identification of the Key Amino Acids of the Interaction Between Fragile Histidine Triad and Replication Protein A

作　　者：张勇[1,2] 于芳[1] 张宏达[1] 绳纪坡[1] 高宁[1] 张鸿声[1,3] 胡宝成[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]福建农林大学农产品品质研究所,福建福州350002 [3]福建农林大学生物农药与化学生物学教育部重点实验室,福建福州350002

出　　处：《生物技术通讯》2008年第6期799-802,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30770651;30670616;30370441);国家重点基础研究发展计划(2005CB522506;2007CB914604);国家"十一五"科技支撑项目(2006BAI23B02)

摘　　要：目的:研究脆性组氨酸三联体(Fhit)对ATR/CHK1通路的影响,在确定Fhit与复制蛋白A(RPA)存在相互作用的基础上鉴定Fhit与RPA相互作用的关键氨基酸残基,为进一步研究Fhit特异的信号通路奠定基础。方法:构建一系列Fhit缺失突变体基因Fhit1～Fhit11及6种Fhit点突变体基因,将这些基因插入含GST基因的原核表达载体中,在大肠杆菌中表达并纯化GST-Fhit1～GST-Fhit11融合蛋白、突变体GST-FhitSIYEEL、GST-FhitIY、GST-FhitEL、GST-FhitF、GST-FhitA,以及GST-FhitD融合蛋白,用GST沉降技术研究Fhit与RPA相互作用的关键氨基酸残基。结果:Fhit蛋白第112～117(SIYEEL)残基可能是Fhit与RPA相互作用的关键区域,而第114(Y)残基可能是Fhit与RPA相互作用的关键氨基酸残基。结论:确定了Fhit与RPA相互作用的关键氨基酸残基,为阐明Fhit在维持基因组完整性方面的机理提供了线索。Objective： To study how fragile histidine triad （Fhit） affects the ATR/CHK1 pathway; the interaction between Fhit and replication protein A（RPA） has been determined, the key amino acids of the interaction will be identified, so as to lay a foundation for studying the specific Fhit signal pathways. Methods： To construct a series of Fhit deleted mutant genes Fhit1-Fhit11 and six point mutant genes, these genes were inserted into the vector containing GST gene. GST-Fhit1 -GST-Fhit11, GST-FhitSIYEEL, GST-FhitIY, GST-FhitEL, GST-FhitF, GST-FhitA and GST-FhitD fusion protein were ex- pressed and purified in E.coli, the key animo acids of the interaction between Fhit and RPA were identified by GST-pull down in vitro. Results： The 112-117 （SIYEEL） animo acids of Fhit protein may be the key domian and Yl14 of Fhit protein may be the kay amino acid for Fhit to interact with RPA by using GST-pull down assays. Conclusion： The kay amino acids of interaction between Fhit and RPA were determined, it provides a clue to elucidate the mechanism for Fhit maintaining genomie integrity.

关 键 词：脆性组氨酸三联体 复制蛋白A 蛋白相互作用 

分 类 号：Q25[生物学—细胞生物学] Q75