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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.
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 / Nc / 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 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. 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 that that higher percentage of the replicating nuclei is either 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 direct interactions with Mcm7. The interaction between M-CSF and Mcm7 promotes both initiation and elongation of DNA replication.