Objective To explore the effect of the interaction between microphage colony-stimulating factor(M-CSF)and minichromosome maintenance protein-7(Mcm7)on DNA replication in HeLa cells.Methods pCMV/nuc/myc,pCMV/nuc/GFP an...Objective To explore the effect of the interaction between microphage colony-stimulating factor(M-CSF)and minichromosome maintenance protein-7(Mcm7)on DNA replication in HeLa cells.Methods pCMV/nuc/myc,pCMV/nuc/GFP and pCMV/nuc/M-CSF vector were stably transfected into HeLa cells by Lipofectamine,respectively.After screening with G418,the expression and localization of M-CSF in HeLa cells were verified by RT-PCR,Western blot and immunofluorescence staining.The statue and interaction between intracellular M-CSF and Mcm7 in HeLa cells was analyzed by co-immunoprecipitation.The effect of the interaction between M-CSF and Mcm7 on DNA replication was analyzed by a mammalian cell or cell-free DNA replication system in vitro.Results The results indicated that the M-CSF-transfected HeLa cells stably express both M-CSF mRNA and protein,and that M-CSF protein is located to the nuclei of HeLa cells mentioned above.To further analyze the status and interaction between intracellular M-CSF and Mcm7,the Mcm7 from HeLa cells was precipitated with anti-Mcm7 antibody and followed by Protein A/G PLUS agarose.The precipitation was blotted with anti-M-CSF monoclonal antibody.The results show that M-CSF was coprecipitated with Mcm7,so intracellular M-CSF existed in Mcm7-bound state.The DNA replication experiments reveal that a higher percentage of the replicating nuclei is present either in unsynchronized or in both synchronized G1 and S phase M-CSF-transfected HeLa cells,compared with both pCMV/nuc-transfected and un-transfected HeLa cells,which suggests that interaction between M-CSF and Mcm7 promote both the initiation and elongation of DNA replication.Conclusions M-CSF directly interacts with Mcm7.The interaction between M-CSF and Mcm7 promotes both the initiation and elongation of DNA replication.展开更多
文摘Objective To explore the effect of the interaction between microphage colony-stimulating factor(M-CSF)and minichromosome maintenance protein-7(Mcm7)on DNA replication in HeLa cells.Methods pCMV/nuc/myc,pCMV/nuc/GFP and pCMV/nuc/M-CSF vector were stably transfected into HeLa cells by Lipofectamine,respectively.After screening with G418,the expression and localization of M-CSF in HeLa cells were verified by RT-PCR,Western blot and immunofluorescence staining.The statue and interaction between intracellular M-CSF and Mcm7 in HeLa cells was analyzed by co-immunoprecipitation.The effect of the interaction between M-CSF and Mcm7 on DNA replication was analyzed by a mammalian cell or cell-free DNA replication system in vitro.Results The results indicated that the M-CSF-transfected HeLa cells stably express both M-CSF mRNA and protein,and that M-CSF protein is located to the nuclei of HeLa cells mentioned above.To further analyze the status and interaction between intracellular M-CSF and Mcm7,the Mcm7 from HeLa cells was precipitated with anti-Mcm7 antibody and followed by Protein A/G PLUS agarose.The precipitation was blotted with anti-M-CSF monoclonal antibody.The results show that M-CSF was coprecipitated with Mcm7,so intracellular M-CSF existed in Mcm7-bound state.The DNA replication experiments reveal that a higher percentage of the replicating nuclei is present either in unsynchronized or in both synchronized G1 and S phase M-CSF-transfected HeLa cells,compared with both pCMV/nuc-transfected and un-transfected HeLa cells,which suggests that interaction between M-CSF and Mcm7 promote both the initiation and elongation of DNA replication.Conclusions M-CSF directly interacts with Mcm7.The interaction between M-CSF and Mcm7 promotes both the initiation and elongation of DNA replication.
