Induction and repair of DNA double-strand breaks in human cells: dephosphorylation of histone H2AX and its inhibition by calyculin A.
Phosphorylation of histone H2AX at serine 139 (gamma-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 gamma rays in MRC-5 human fibroblasts was studied. The number of gamma-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 gamma-H2AX foci occurred. This indicates that the number of gamma-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 gamma-H2AX dephosphorylation and DSB rejoining are independent phenomena. We found that the maintenance of the phosphate group at Ser 139 in gamma-H2AX does not represent an obstacle for DSB rejoining. Preliminary experiments performed with 62 MeV/nucleon carbon ions have shown a longer persistence of gamma-H2AX foci with respect to gamma rays, consistent with the induction of damage that is more severe and difficult to repair.
Antonelli
Francesca
F
Istituto Superiore di Sanità, Technology and Health Department, 00161 Rome, Italy. francesca.antonelli@iss.it
Belli
Mauro
M
Cuttone
Giacomo
G
Dini
Valentina
V
Esposito
Giuseppe
G
Simone
Giustina
G
Sorrentino
Eugenio
E
Tabocchini
Maria Antonella
MA
D004249:DNA Damage; D004260:DNA Repair; D016521:Electrophoresis, Gel, Pulsed-Field; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology*; D010766:Phosphorylation
C468783:H2AX protein, human; D006657:Histones; D008387:Marine Toxins; D010080:Oxazoles; C059041:calyculin A