Effect Of The Interaction Between M-CSF And MCM7 On DNA Replication

ObjectivesTo establish a cell line that stably express M-CSF in the nucleus of HeLa cells. To verify M-CSF bound to Mcm7. To explore the effect of the interaction between M-CSF and Mcm7 on DNA replication.MethodspCMV/nuc/myc, pCMV/nuc/GFP and pCMV/nuc/M-CSF vectors 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 status 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.ResultsThe results indicate that the M-CSF-transfected HeLa cells express both M-CSF mRNA and protein, and that M-CSF protein is located to the nuclei of HeLa cells mentioned above, which suggests a cell line stably expressing M-CSF is established. To further analyze the status and interaction between intracellular M-CSF and Mcm7, the Mcm7 from HeLa cells was precipitated with anti-Mcm7 monoclonal 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 (40.134% vs 21.23% vs 20.53%) or in both synchronized G1 (87.95% vs 63.84% vs 62.34%) and S phase (90.09% vs 71.32% vs 70.54%) M-CSF-transfected HeLa cells, compared with both pCMV/nuc-transfected and un-transfected HeLa cells, which suggests that the interaction between M-CSF and Mcm7 promotes both the initiation and elongation of DNA replication.ConclusionA cell line that expresses stably nuclear M-CSF was established. M-CSF directly interacts with Mcm7. The interaction between M-CSF and Mcm7 promotes both the initiation and elongation of DNA replication.