【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。更多还原

【Abstract】 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 expressed 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 Y114 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 genomic integrity.更多还原