TGFß1 Cell Cycle Arrest Is Mediated by Inhibition of MCM Assembly in Rb-Deficient Conditions.

Transforming growth factor ß1 (TGFß1) is a potent inhibitor of cell growth that targets gene-regulatory events, but also inhibits the function of CDC45-MCM-GINS helicases (CMG; MCM, Mini-Chromosome Maintenance; GINS, Go-Ichi-Ni-San) through multiple mechanisms to achieve cell-cycle arrest. Early in G1, TGFß1 blocks MCM subunit expression and suppresses Myc and Cyclin E/Cdk2 activity required for CMG assembly, should MCMs be expressed. Once CMGs are assembled in late-G1, TGFß1 blocks CMG activation using a direct mechanism involving the retinoblastoma (Rb) tumor suppressor. Here, in cells lacking Rb, TGFß1 does not suppress Myc, Cyclin E/Cdk2 activity, or MCM expression, yet growth arrest remains intact and Smad2/3/4-dependent. Such arrest occurs due to inhibition of MCM hexamer assembly by TGFß1, which is not seen when Rb is present and MCM subunit expression is normally blocked by TGFß1. Loss of Smad expression prevents TGFß1 suppression of MCM assembly. Mechanistically, TGFß1 blocks a Cyclin E-Mcm7 molecular interaction required for MCM hexamer assembly upstream of CDC10-dependent transcript-1 (CDT1) function. Accordingly, overexpression of CDT1 with an intact MCM-binding domain abrogates TGFß1 arrest and rescues MCM assembly. The ability of CDT1 to restore MCM assembly and allow S-phase entry indicates that, in the absence of Rb and other canonical mediators, TGFß1 relies on inhibition of Cyclin E-MCM7 and MCM assembly to achieve cell cycle arrest. IMPLICATIONS: These results demonstrate that the MCM assembly process is a pivotal target of TGFß1 in eliciting cell cycle arrest, and provide evidence for a novel oncogenic role for CDT1 in abrogating TGFß1 inhibition of MCM assembly.

Destabilization of the MiniChromosome Maintenance (MCM) complex modulates the cellular response to DNA double strand breaks.

DNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is replicated only once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both the initiation and elongation of DNA replication. Although only two DNA helicase activities are necessary to establish a bidirectional replication fork from each replication origin, a large excess of MCM complexes is amassed and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most are displaced from the DNA during the S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of a reduction in MCM complex. The alteration of MCM proteins induced a change in the activation of key factors of the DDR in response to Etoposide treatment. Etoposide-induced DNA damage affected the phosphorylation of γ-H2AX, CHK1 and CHK2 without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we identified a decrease in both HR and NHEJ associated with a decrease in MCM complex.

Minichromosome Maintenance Complex is Required for Checkpoint Kinase 2 Chromatin Loading and its Phosphorylation to DNA Damage Response in SCC-4 Cells.

Checkpoint kinase 2 (Chk2) is a significant mediator of diverse responses to DNA damage. The present study was aimed to identify possible interactive proteins of Chk2 and try to clarify the underlying mechanism regarding Chk2 chromatin loading and its phosphorylation to DNA damage response in oral squamous cell carcinoma (OSCC). Differently tagged Chk2 and minichromosome maintenance (MCM) complex (MCM2, MCM3, MCM5, and MCM6) were overexpressed into SCC-4 cells. After 48 h of transfection cell fractionation was performed to localize proteins. In addition, immunoreactive species were detected by immunoprecipitation (IP) and immunoblot (IB) analysis, and protein-protein interaction between Chk2 and MCM complex was ensured by glutathione S-transferase (GST) pull-down assay. Expression of MCM2 and MCM6 was downregulated by small interfering RNA (siRNA), and the chromatin and non-chromatin fraction were analyzed. The expression of Chk2 phosphorylation (pT68-Chk2) was measured after administration of different dosages of siMCM2 (0.5 µg, 1 µg, and 2.5 µg) and camptothecin (CPT). Our results showed that Chk2 directly interacts with MCM2, MCM3, MCM5, and MCM6 in SCC-4 cells. Downregulation of MCM2 and MCM6 markedly reduced Chk2 chromatin fraction, and downregulation of MCM2 decreased the expression of pT68-Chk2 to DNA damage response in a dose manner. Our results suggest that the interaction between Chk2 and MCM complex is required for Chk2 chromatin loading and its phosphorylation to DNA damage response in SCC-4 cells.