The Mechanism Of Fhit Mutant In DNA Damage Response

Fragile histidine triad (Fhit) gene, the first cloned and identified common fragile site gene, spans FRA3B Fragile area and is located in 3p14.2. Fhit is confirmed to be an tumor suppressor by using cell biology, oncomolecularbiology, transgenic and gene knock-out technologies, it is involved in tumorigenesis and tumor development. We previously reported that Fhit-/- cells with an over-activated ATR/CHK1 pathway exhibited higher mutation rate and more resistance to multi-DNA damage inducers compared with Fhit+/+ cells. To further study the mechanism how Fhit affects the ATR/CHK1 pathway, we screened the proteins which were related with the ATR/CHK1 pathway by Immunoprecipitation (IP), and the result showed that Fhit can interact with RPA (Replication Protein A, RPA). Then we identified that Fhit I113 and Y114 are the key amino-acid residues for Fhit to interact with RPA. In this study, a series of Fhit mutants were constructed, and Fhit Y114 was identified by GST-pull down as the key amino acid residues for this interaction. We also determined that Fhit affected the ATR/CHK1 pathway through its interaction with RPA.First, Fhit Y114 was mutagenized to Fhit A114, Fhit D114 and Fhit F114 (FhitA, FhitD and FhitF) by PCR, then these mutant genes were inserted into pGEX-KG vector. GST-FhitA, GST-FhitD and GST-FhitF fusion proteins were expressed and purified in E.coli, and GST-pull down method was used to study whether the Fhit mutants can interact with RPA in vitro. Meanwhile three Fhit mutant genes were inserted into eukaryotic expression vector pREP10, and the cell lines which can stably express Fhit mutants were obtained. We identified that the key amino acid residue of the interaction between Fhit and RPA was Fhit Y114 by using immunofluorescence staining, colony forming assay, MTT assay and cell cycle assay. Finally we found that compared with the human cells expressing the wild-type Fhit, the ones expressing the Y114 mutant Fhit showed more resistance to DNA damage inducers and more significant G2 phase arrest following IR (Ion Radiation, IR), which is similar to the phenotype of Fhit deficient cells.In this study, the key amino acid residue for Fhit interacting with RPA was identified, and it was determined that Fhit affected the ATR/CHK1 pathway through its interaction with RPA. These results may give a clue to elucidate the mechanism of Fhit maintaining genomic integrity, and may also conduce to learn the roles Fhit plays in tumorigenesis.