Original Article
The interaction between TopBP1 and MCM complex is essential for TopBP1 foci formation in colon cancer
Abstract
Background: Topoisomerase IIβ-binding protein 1 (TopBP1) and minichromosome maintenance proteins (MCM) have been reported to be core elements for DNA replication and mitosis progression. This study was aimed to investigate the interactions between TopBP1 and MCM complex.
Methods: The recombinant vectors of TopBP1 and MCMs with or without tags were constructed and transfected into HCT116 cells. After chromatin fraction, immunoprecipitation (IP), and Western blotting, in vitro Glutathione S-transferase (GST) pull-down assay, and mass spectrometry (MS) were performed to identify the potential interaction between TopBP1 and MCM complex. Numbers of bleomycin (BLM)-induced TopBP1 foci positive cells were detected by immunocytochemical detection in HCT116 cells transfected with MCMs short hairpin RNA (shRNA).
Results: The results showed that four MCMs (MCM2, 3, 5, and 6) were identified as TopBP1-interacting proteins. In addition, we observed that there was an interaction between endogenous TopBP1 and MCM2 and MCM6 proteins. We successfully suppressed the expression of MCM2 and MCM6 using shRNAs. Then a clear reduction of TopBP1 level in chromatin compartment was detected. Bleomycin (BLM) -induced TopBP1 foci positive cells were identified. However, the percentage of TopBP1 positive cells was significantly reduced after silencing of MCM2 and MCM6.
Conclusions: These results suggested that TopBP1 interacted with MCM2 and MCM6 and that MCM2 and MCM6 were essential factors for TopBP1 foci formation.
Methods: The recombinant vectors of TopBP1 and MCMs with or without tags were constructed and transfected into HCT116 cells. After chromatin fraction, immunoprecipitation (IP), and Western blotting, in vitro Glutathione S-transferase (GST) pull-down assay, and mass spectrometry (MS) were performed to identify the potential interaction between TopBP1 and MCM complex. Numbers of bleomycin (BLM)-induced TopBP1 foci positive cells were detected by immunocytochemical detection in HCT116 cells transfected with MCMs short hairpin RNA (shRNA).
Results: The results showed that four MCMs (MCM2, 3, 5, and 6) were identified as TopBP1-interacting proteins. In addition, we observed that there was an interaction between endogenous TopBP1 and MCM2 and MCM6 proteins. We successfully suppressed the expression of MCM2 and MCM6 using shRNAs. Then a clear reduction of TopBP1 level in chromatin compartment was detected. Bleomycin (BLM) -induced TopBP1 foci positive cells were identified. However, the percentage of TopBP1 positive cells was significantly reduced after silencing of MCM2 and MCM6.
Conclusions: These results suggested that TopBP1 interacted with MCM2 and MCM6 and that MCM2 and MCM6 were essential factors for TopBP1 foci formation.
