A Critical Role for Histone H2AX in Recruitment of Repair Factors to Nuclear Foci After DNA Damage

Curr Biol. 2000 Jul 27-Aug 10;10(15):886-95. doi: 10.1016/s0960-9822(00)00610-2.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Androstadienes / pharmacology
  • BRCA1 Protein / genetics
  • BRCA1 Protein / metabolism
  • Bromodeoxyuridine / pharmacology
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • DNA Damage*
  • DNA Repair Enzymes*
  • DNA Repair*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Enzyme Inhibitors / pharmacology
  • Gamma Rays
  • Histones / metabolism*
  • Humans
  • Lasers
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphorylation
  • Rad51 Recombinase
  • Tumor Cells, Cultured
  • Ultraviolet Rays
  • Wortmannin

Substances

  • Androstadienes
  • BRCA1 Protein
  • DNA-Binding Proteins
  • Enzyme Inhibitors
  • Histones
  • Phosphoinositide-3 Kinase Inhibitors
  • RAD51 protein, human
  • Rad51 Recombinase
  • Rad50 protein, human
  • DNA Repair Enzymes
  • Bromodeoxyuridine
  • Wortmannin