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Purification of Ubiquitinated p53 Proteins from Mammalian Cells
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A Direct Interaction Between P53-Binding Protein 1 and Minichromosome Maintenance Complex in Hepg2 Cells

Yong Chen1,2, Chengyin Weng1, Hui Zhang2

  • 1Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, China.

Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology
|July 11, 2018
PubMed
Abstract

Insights

p53-binding protein 1 (53BP1) directly interacts with minichromosome maintenance (MCMs) proteins in liver cancer cells. This interaction is crucial for 53BP1

Area of Science:

  • Molecular biology
  • Cancer research
  • Hepatocellular carcinoma (HCC) studies

Background:

  • Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality globally.
  • Targeting DNA damage repair mechanisms in cancer cells presents a promising therapeutic strategy.
  • Investigating protein interactions involved in DNA repair is key to understanding cancer progression.

Purpose of the Study:

  • To explore the interaction between p53-binding protein 1 (53BP1) and minichromosome maintenance (MCMs) proteins.
  • To elucidate the role of this interaction in DNA damage response within human hepatoma HepG2 cells.

Main Methods:

  • Constructed recombinant vectors for tagged 53BP1 and MCMs, transfected into HepG2 cells.
  • Utilized immunoprecipitation (IP) and mass spectrometry (MS) to identify protein interactions.
  • Performed glutathione S-transferase (GST) pull-down assays for direct interaction validation.
  • Examined 53BP1 chromatin fraction and foci formation after MCM2/MCM6 knockdown via shRNAs under DNA damage conditions.

Main Results:

  • MCM2/3/5/6 proteins were immunoprecipitated with HA-tagged 53BP1 in HepG2 cell nuclei.
  • GST pull-down assays confirmed a direct interaction between 53BP1 and the MCMs complex.
  • Down-regulation of MCM2 or MCM6 increased non-chromatin 53BP1 levels while decreasing chromatin-bound 53BP1.
  • Knockdown of MCM2 or MCM6 significantly inhibited 53BP1 foci formation following bleomycin-induced DNA damage (P < 0.01).

Conclusions:

  • A direct interaction exists between 53BP1 and MCMs in hepatoma cells.
  • This interaction is essential for the proper chromatin localization and foci formation of 53BP1.
  • Findings contribute to understanding DNA repair mechanisms in HCC and potential therapeutic targets.
Keywords:
CarcinomaChromatinHepatocellularMinichromosome Maintenance ProteinsTumor Suppressor Protein p53

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