巨噬细胞集落刺激因子及微小染色体维持蛋白7相互作用及其对DNA复制影响.pdfVIP

Effect of the Interaction between M-CSF and MCM7 on DNA Replication ABSTRACT Objectives To 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. Methods pCMV/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. Results The 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