A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks

Seung Yun Lee, Ji Hye Park, Sungsu Kim, Eun Jung Park, Yungdae Yun, Jongbum Kwon

Research output: Contribution to journalArticlepeer-review

68 Scopus citations

Abstract

Double-strand breaks (DSBs) of chromosomal DNA trigger the cellular response that activates the pathways for DNA repair and cell-cycle checkpoints, and sometimes the pathways leading to cell death if the damage is too severe to be tolerated. Evidence indicates that, upon generation of DNA DSBs, many nuclear proteins that are involved in DNA repair and checkpoints are recruited to chromatin around the DNA lesions. In the present study we used a proteomics approach to identify DNA-damage-induced chromatin-binding proteins in a systematic way. Two-dimensional gel analysis for protein extracts of chromatin from DNA-damage-induced and control HeLa cells identified four proteins as the candidates for DNA-damage-induced chromatin-binding proteins. MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis identified these proteins to be NPM (nucleophosmin), hnRNP (heterogeneous nuclear ribonucleoprotein) Cl, hnRNP C2 and 37-kDa laminin-receptor pre-cursor, and the identity of these proteins was further confirmed by immunoblot analysis with specific antibodies. We then demonstrated with chromatin-binding assays that NPM and hnRNP C1/C2, the abundant nuclear proteins with pleiotropic functions, indeed bind to chromatin in a DNA-damage-dependent manner, implicating these proteins in DNA repair and/or damage response. Immunofluorescence experiments showed that NPM, normally present in the nucleoli, is mobilized into the nucleoplasm after DNA damage, and that neither NPM nor hnRNP C1/C2 is actively recruited to the sites of DNA breaks. These results suggest that NPM and hnRNP C1/C2 may function at the levels of the global context of chromatin, rather than by specifically targeting the broken DNA.

Original languageEnglish
Pages (from-to)7-15
Number of pages9
JournalBiochemical Journal
Volume388
Issue number1
DOIs
StatePublished - May 15 2005

Keywords

  • Chromatin
  • DNA damage
  • Heterogeneous nuclear ribo-nucleoprotein (hnRNP)
  • Nucleophosmin
  • Proteomics
  • Radiation

Fingerprint

Dive into the research topics of 'A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks'. Together they form a unique fingerprint.

Cite this