The Effect Of BACH1 Interaction With BTF On DNA Damage Repair

Breast cancer has become the highest incidence of malignant tumors in the world with increased morbidity and mortality rate.In China,malignant tumor has not only been a threat to public health,but also a serious social problem affecting the quality of people’s lives and restricting the economic development.Therefore,understanding the underlying mechanism of tumorigenesis,development,recurrece and metastasis is the key to develop new anti-tumor drugs.BACH1(BRIP1,FANCJ)encodes a protein belonging to the RecQ DEAH helicase family.Aberrations in BACH1 have been mainly associated with the occurrence and development of breast cancer(BC),ovarian cancer,and type J Fanconi anemia.It interacts with the C-terminal BRCT domain of BRCA1 and participates in the repair of DNA damage and the inhibition of tumorigenesis with the complexes of CtIP and Abraxas / RAP80 in the BRCT domain.DNA damage repair system plays a key role in maintaining genomic stability and suppressing oncogenesis.The mutation or abnormal expression of genes involved in DNA damage response(DDR)may cause tumorigenesis.BACH1,an important mediator in DNA damage response,plays an important role in regulating DNA replication stress response,homologous recombination repair(HR)and cross-linking repair.Mutations in BACH1 will affect the intracellular function of DNA damage repair and lead to the evolution and progression in malignant tumors.First of all,we identified BTF as a new interacting protein of BACH1 through a series of assays,such as protein affinity purification,mass spectrometry and immunoprecipitation.Furthermore,the interaction between BACH1 and BTF depended on the D9 domain of BACH1 and the D7 domain of BTF.Deletion of the D9 domain of BACH1 and the D7 domain of BTF could both abrogate the interaction between BACH1 and BTF.DNA damage induced the increase of the expression levels of BACH1 and BTF,while the interaction between the two proteins is also increased.After inducing DNA damage,we found that BACH1,BTF and γH2AX all formed Foci at the same DNA damage site by immunofluorescence assay,indicating that BACH1 and BTF could colocalize in DNA damage repair.Next,we induced DNA damage in living cells by laser microirradiation.Imaging results showed that the deletion of the D9 domain of BACH1 interacting with BTF did not affect its recruitment at the site of DNA damage,whereas BTF lacked of the D7 domain that could not interact with BACH1 affected its involvement in the DNA damage repair process,indicating that the BACH1 protein is located upstream of the BTF protein in the DNA damage repair pathway.We knocked down the expression of BACH1 and BTF in HEK293 T cells by siRNA and then induced the DNA damage in living cells through laser microirradiation.The results of imaging showed that knocking down BACH1 affected the recruitment of BTF at the DNA damage sites,knocking down BTF did not affect BACH1 at the DNA damage sites.The result proved that BACH1 is the upstream protein of BTF in the DNA damage repair pathway futher.In addition,we also studied the effect of BACH1 and BTF on cell proliferation,and found that both BACH1 knockdown and BTF knockdown have a negative effect on cell proliferation.Knocking down the two proteins at the same time has a more significantly influence on cell proliferation,indicating that these two proteins also play an important role in cell proliferation.