Study On The Mechanism Of Unwinding Under DNA Cross-linking Damage

DNA is the most basic process in an organism.DNA molecules are attacked by many elements every day.These attacks are caused by internal and external factors.These attacks cause changes in DNA structure,which often lead to genetic damage in the organism.When the DNA damage repair mechanism fails to completely repair the damage,it will Causes cancer or death of cells.DNA interstrand cross-linking(ICLs)is a highly toxic gene damage,which will hinder the transcription and replication of cells and the completion of cell cycle.One of the repair pathways that exist in cells is a specific ICLs-sensitive signaling pathway,known as the fanconi anemia signaling pathway,which can mediate a variety of repair pathways in response to ICLs damage.So far,17 proteins related to fanconi anemia have been found,all of which are involved in the repair of ICLs damage.FANCM protein,in addition to participating in the signaling pathway of fanconi anemia,plays a key role in the repair of ICLs itself.The translocase activity of the FANCM/MHF complex protein mediates the main mode of ICLs repair--replication fork tranverse--which promotes the replication fork traverse the ICLs damage site from upstream to downstream DNA.MCM helicase is the only replication-related six-membrance cyclase found in mammals.It only activates the helicase activity after binding to Cdc45 and GINS protein and filling the gap of the six-membrance cyclase to cause conformational changes,and vice versa.In this study,Dig-TMP was used to investigate the mechanism of DNA cross-linking damage.FANCM protein was taken as the main object of study,and the relationship between ICLs-related translocase FANCM and the helicase MCM necessary for unwinding and the open-ring mechanism of CMG complex when it encountered ICLs were studied through in vivo protein-protein interaction verification technology – Co-immunoprecipitation and proximity ligation assay.We found that there was an interaction between FANCM protein and MCM protein and Cdc45 protein in the CMG complex,which was ATR dependent.The phosphorylated MCM2 could interact with FANCM protein.To our surprise,GINS complex protein dropped from the CMG complex.Further study found that FANCM protein and GINS protein could not interact with MCM protein and Dig-TMP at the same time.This indicates that when replication forks encounter ICLs,FANCM protein will be recruited to ICLs site and activate ATR.MCM protein phosphorylated by ATR can interact with FANCM protein and squeeze out GINS complex protein,open the closed ring of CMG,promote the CM complex to traverse ICLs from upstream to downstream DNA,and unwind the DNA there.