RECQ4 Interacts With CST Complex To Restart The Stalled Replication Fork

Objective: In mammals,telomeres consist of repeated DNA sequences of TTAGGG and the telomere-binding protein Shelterin and play an important role in maintaining chromosome integrity,preventing chromosome fusion,and maintaining genomic stability.However,unlike most genomic DNA,the repeat sequence of telomere DNA tends to form high-level structures,such as T-loop and G-quadruplex(G4),especially since the existence of G4 structure,DNA double strand cannot be used as a template for DNA replication.In order for the telomeres to replicate properly,the G4 structure needs to be turned on.We previously found that another telomere binding complex,CST,has the function of binding G4,and its absence contributes to the accumulation of G4 structure in vivo and causes the replication fork stalling of telomeres.We found that RECQ4,a member of the RECQ helicase family,plays an important role in DNA replication and the unwinding of DNA higher structures.In this study,on the one hand,we will discuss and define RECQ4 telomeric biology function,expounds the telomere replication and the role of telomere DNA damage.On the other hand,we will explore and identify molecular basis that RECQ4 interact with CST restart blocked replication forks of telomeres.That will help us to understand comprehensively the mechanisms of telomere DNA replication in the cancer foundation,and provide a new target and plan to suppress telomere replication as the means of tumor therapy.Methods:(1)Mass spectrometry and Immunoprecipitation(CO-IP)technique was used to verified the interaction between RECQ4 and CST complex.Coimmunofluorescence(CO-IF)was used to verify the interaction basis of RECQ4 and CST on telomeres.(2)The most common methods to verify the function of a gene are knockout,knockout and overexpression.sh RNA and si RNA technique were used to construct the stable knock-down and instantaneous knock-down RECQ4 cell lines.Flow cytometry and Ed U incorporation technique were used to test the effect of RECQ4 on cell cycle regulation and genomic DNA replication;Immunofluorescence(IF)? micronucleus test and cell proliferation toxicity test were used to analyze the degree of DNA damage?genomic toxicity and cell proliferation.(3)Fluorescence in situ hybridization(FISH)and immunofluorescence in situ hybridization(IF-FISH)were used to analyze the telomere function and telomere specific DNA damage after RECQ4 knock-down.(4)Co-immunofluorescence,fluorescence in situ hybridization(FISH)and immunofluorescence-fluorescence in situ hybridization(IF-FISH)were used to detect the role of the interaction between RECQ4 and CST telomere.DNA Fiber and Fiber-FISH technique were used to analyze the role of RECQ4 and CST in the stalled replication fork and definite the regulation mechanism of their interaction on the restart of stalled replication fork in genome and telomere.Results:(1)Firstly,the results of mass spectrometry and immunoprecipitation showed that RECQ4 could interact with CST.Co-immunofluorescence showed that RECQ4 could be located on telomeres,and its location on telomeres depended on STN1.(2)The RT-PCR and western blot show the effectively reduced efficiency when we used the sh RNA or si RNA constructing the RECQ4 knock-down cell lines.Flow cytometry and Ed U incorporation data show the cell cycle was changed and the DNA replication efficiency of the genome was reduced after RECQ4 knock-down.Immunofluorescence and micronucleus test results showed that after RECQ4 knock-down,the level of DNA damage and the number of micronucleus increased.The cell proliferation-toxicity showed that after RECQ4 knockdown,the cells are more sensitive to DNA replication block reagent and the proliferation activity of cells decreased.(3)Fluorescence in situ hybridization(FISH)results showed that telomere loss(SFE)and multi-telomere signal(MTS)significantly increased after RECQ4 knock-down.Immunofluorescence-fluorescence in situ hybridization results(IF-FISH)showed that after RECQ4 reduction,the level of DNA damage of telomeres also significantly increased.These results all indicate the effect of RECQ4 on telomere maintenance.(4)Fluorescence in situ hybridization showed that after RECQ4 and STN1 were knocked down separately,the proportion of telomere abnormalities increased.Although there was an increased telomeric abnormalities after RECQ4 and STN1 knocked down simultaneously,no additional increase effect was observed.Similarly,the results of immunofluorescence-fluorescence in situ hybridization showed that the number of G4 and the proportion of G4 in the telomere increased significantly after RECQ4 and STN1 were knocked down separately,and there was no additional increase in the number of G4 when RECQ4 and STN1 were knocked down simultaneously.These results indicate that RECQ4 interact with CST and locate on the telomeres to solve the telomere replication problem in the same pathway.(5)DNA Fiber results showed that when the PDS treatment cells caused replication pressure,the efficiency of DNA replication restart was reduced in the RECQ4 and STN1 knockout cells.Meanwhile,in the RECQ4 and STN1 double-knocked down cells,the efficiency of DNA replication restart also decreased,but there was no trend of additional reduction.In addition,we detected the state of telomeres replication forks through Fiber-FISH,and the results showed that in the RECQ4 and STN1 knockout cells,the number of stalled replication forks and replication forks that could not fully replicate increased,indicating that RECQ4 and CST restarted the blocked replication forks on the genome and telomeres through interaction.However,when RECQ4 and STN1 were knock-down at the same time,the abnormal replication fork showed no additional trend.These results suggest that the interaction of RECQ4 and CST may play the role of restarted blocked replication forks on the genome and telomeres in a same signaling pathway.Conclusion: We found that the helicase protein RECQ4 interacts with the CST complex,and RECQ4 can be located on the telomeres in a CST-dependent manner.Nest,we verified the function of RECQ4.It was found that RECQ4 knockdown affected cell cycle and genomic DNA replication,caused DNA damage and chromosome abnormalities,and increased cell sensitivity to DNA replication inhibitory drugs,leading to cell proliferation inhibition.In addition,RECQ4 also plays an important role in the maintenance of telomere function.When RECQ4 is reduced,telomere loss and multitelomere signal in the cell are significantly increased,and the level of telomere specific DNA damage is also increased.Importantly,we found that RECQ4 and CST interactions may play a role in the same signaling pathway to open the G4 structure and maintain normal telomeres replication.Finally,through DNA Fiber and Fiber-FISH analysis,RECQ4 can restart the blocked replication fork through interaction with CST to maintain the normal replication of genome and telomere DNA.