A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.
BACKGROUND
The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 1-3 minutes of DNA damage and forms foci at break sites.


RESULTS
In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (gamma-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with gamma-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent gamma-H2AX formation, indicating that gamma-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for gamma-H2AX in DNA repair.


CONCLUSIONS
The pattern of gamma-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the gamma-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.
Paull
T T
TT
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0540, USA.
Rogakou
E P
EP
Yamazaki
V
V
Kirchgessner
C U
CU
Gellert
M
M
Bonner
W M
WM
D017766:Acid Anhydride Hydrolases; D000730:Androstadienes / Q000494:pharmacology; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005720:Gamma Rays; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007834:Lasers; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051135:Rad51 Recombinase; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays; D000077191:Wortmannin
D017766:Acid Anhydride Hydrolases; D000730:Androstadienes; D019313:BRCA1 Protein; D001973:Bromodeoxyuridine; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006657:Histones; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051135:Rad51 Recombinase; D000077191:Wortmannin; C495153:RAD51 protein, human; C112158:RAD50 protein, human