Cloning And Eukaryotic Expression Of MCM5 And Confirmation Of MCM5 As A New P53 Interactor

P53, a forever intriguing and mysterious character on the stage of cell life, has long been known as"cellular gatekeeper"or"guardian of the genome"for its pivotal roles in coordinating and integrating the cellular responses to a variety of external stress stimuli. P53 serves as a node to analyze and response to different kinds and levels of stress by initiating various downstream signaling pathways, such as apop- -tosis, cell cycle arrest, senescence, DNA repair, changes in cell metabolism state or autophagy.P53 undergoes different types of post-translational modifications which trigger different cellular effects in response to various kinds of stress factors. P53 consists of the N-terminal proline-rich transactivation domain, the DNA-binding domain, followed by the tetramerization domain and C-terminal domain. The major post-translational modifications to p53 in response to different stresses occur at theN-terminus of the molecule, a region responsible for the transactivation activity of p53 and where can interact with MDM2. P53 is responsible for the transcriptional activation of a series of proteins involved in cell cycle control, apoptosis, senescence and so on.P53-controlled transactivation of target genes is an essential feature of each stress response pathway, although some effects of p53 may be independent of transcription (Vousden and Lane, 2007; Marchenko and Moll, 2007) . Besides MDM2( mouse double minute 2), the stability of p53 is also influenced by a series of other interacting proteins.Here in an attempt to seek for novel p53 interacting proteins, we employed fusion protein GST-p53N60 which includes the sixty amino acids from the N-teminus of full length homo p53 as bait to perform GST pull-down assay with HeLa cell lysate. By the way of mass spectrometry analysis, we fortunately obtained a potential candidate called MCM5. MCM5, acting as a DNA replication licensing factor, is a member of the evolutionarily conserved eukaryotic MCM (minichromosome maintainance) family. Full-length homo MCM5 gene was generated by PCR from a HeLa cDNA library and subcloned into two eukaryotic expression vectors pEGFP-C1 and p3*flag-myc-CMV-24. Then GST pull-down and coimmunopricipitation were adopted to verify that p53 can truly interact with MCM5, one of the regulatory subunits of MCM2-7 helicase.