陆军军医大学学报 (Aug 2023)

DNA damage repair protein PARP1 poly (ADP-ribosyl) ates mitotic protein BUB3 regulates mitosis in HeLa cells

  • YANG Xue,
  • YANG Xue,
  • YANG Xue,
  • XU Bo,
  • XU Bo,
  • XU Bo

DOI
https://doi.org/10.16016/j.2097-0927.202305002
Journal volume & issue
Vol. 45, no. 16
pp. 1682 – 1692

Abstract

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Objective To investigate the role and molecular mechanism of poly (ADP-ribose) polymerase 1 (PARP1) in mitosis of HeLa cells. Methods Flow cytometry, immunofluorescent assay and live cell imaging were used to detect the effect of PARP1 on mitosis of HeLa cells. Chromosome karyotype analysis and immunofluorescent assay were used to detect the effect of PARP1 on chromosome stability. Western blotting and co-immunoprecipitation assay were used to analyze the protein expression and enzyme activity of PARP1 in HeLa cells during mitosis. Proteome mass spectrometry was used to identify proteins that interacted specifically with PARP1 in mitosis, which was verified by co-immunoprecipitation and poly(ADP-ribosyl)ation assay. Results Flow cytometry and immunofluorescence staining showed that the response of HeLa cells to nocodazole-induced mitotic arrest was significantly decreased after genetic manipulation (knockdown) or pharmacological inhibition (olaparib) of PARP1 (P < 0.05). The results of live cell imaging showed the mean mitotic time of HeLa cells was shortened after the knockdown of PARP1(P < 0.01). Chromosome karyotype analysis and immunofluorescence staining showed that the proportion of aneuploid and multipolar spindle cells was increased significantly after the knockdown of PARP1 (P < 0.05). Western blotting showed no significant difference in the protein level of PARP1 during the process of mitosis, while the co-immunoprecipitation assay showed the enzyme activity was increased significantly. Proteome mass spectrometry and co-immunoprecipitation assay showed that PARP1 specifically interacted with BUB3, a major component of the mitotic checkpoint complex (MCC), and BUB3 is poly(ADP-ribosyl)ated in mitosis. Conclusion DNA damage repair protein PARP1 may regulate the mitosis of HeLa cells and maintain its chromosome stability by upregulating enzyme activity and poly(ADP-ribosyl)ating mitotic BUB3.

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