Induction and repair of DNA double-strand breaks in human cells: Dephosphorylation of histone H2AX and its inhibition by calyculin A

Francesca Antonelli, Mauro Belli, Giacomo Cuttone, Valentina Dini, Giuseppe Esposito, G. Simone, Eugenio Sorrentino, Maria Antonella Tabocchini

Research output: Contribution to journalArticle

Abstract

Phosphorylation of histone H2AX at serine 139 (γ-H2AX) represents one of the earliest steps in DNA DSB signaling and repair, but the mechanisms of coupling this histone modification to DSB processing remain to be established. In this work, H2AX phosphorylation-dephosphorylation kinetics induced by low doses of γ rays in MRC-5 human fibroblasts was studied. The number of γ-H2AX foci increased rapidly, with the maximum reached 20 min after irradiation. Using calyculin A, a protein phosphatase inhibitor, no significant dephosphorylation was found in this time. At longer times, no further induction of γ-H2AX foci occurred. This indicates that the number of γ-H2AX foci scored at 20 min can be used as representative of the initial number of DSBs. Pulsed-field gel electrophoresis (PFGE) was also used to determine whether calyculin A-mediated inhibition of γ-H2AX dephosphorylation and DSB rejoining are independent phenomena. We found that the maintenance of the phosphate group at Ser 139 in γ-H2AX does not represent an obstacle for DSB rejoining. Preliminary experiments performed with 62 MeV/nucleon carbon ions have shown a longer persistence of γ-H2AX foci with respect to γ rays, consistent with the induction of damage that is more severe and difficult to repair.

Original languageEnglish
Pages (from-to)514-517
Number of pages4
JournalRadiation Research
Volume164
Issue number4 II
DOIs
Publication statusPublished - Oct 1 2005

ASJC Scopus subject areas

  • Biophysics
  • Radiation
  • Radiology Nuclear Medicine and imaging

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