BRCT domain interactions with phospho-histone H2A target Crb2 to chromatin at double-strand breaks and maintain the DNA damage checkpoint

Mol Cell Biol. 2010 Oct;30(19):4732-43. doi: 10.1128/MCB.00413-10. Epub 2010 Aug 2.

Abstract

Relocalization of checkpoint proteins to chromatin flanking DNA double-strand breaks (DSBs) is critical for cellular responses to DNA damage. Schizosaccharomyces pombe Crb2, which mediates Chk1 activation by Rad3(ATR), forms ionizing radiation-induced nuclear foci (IRIF). Crb2 C-terminal BRCT domains (BRCT(2)) bind histone H2A phosphorylated at a C-terminal SQ motif by Tel1(ATM) and Rad3(ATR), although the functional significance of this interaction is controversial. Here, we show that polar interactions of Crb2 serine-548 and lysine-619 with the phosphate group of phospho-H2A (γ-H2A) are critical for Crb2 IRIF formation and checkpoint function. Mutations of these BRCT(2) domain residues have additive effects when combined in a single allele. Combining either mutation with an allele that eliminates the threonine-215 cyclin-dependent kinase phosphorylation site completely abrogates Crb2 IRIF and function. We propose that cooperative phosphate interactions in the BRCT(2) γ-H2A-binding pocket of Crb2, coupled with tudor domain interactions with lysine-20 dimethylation of histone H4, facilitate stable recruitment of Crb2 to chromatin surrounding DSBs, which in turn mediates efficient phosphorylation of Chk1 that is required for a sustained checkpoint response. This mechanism of cooperative interactions with the γ-H2A/X phosphate is likely conserved in S. pombe Brc1 and human Mdc1 genome maintenance proteins.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Binding Sites / genetics
  • Cell Cycle / genetics
  • Cell Cycle / physiology
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Chromatin / genetics
  • Chromatin / metabolism*
  • DNA Breaks, Double-Stranded*
  • DNA Damage
  • DNA Repair
  • Histones / genetics
  • Histones / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Lysine / genetics
  • Lysine / metabolism
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutation
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Protein Binding
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Serine / genetics
  • Serine / metabolism

Substances

  • Cell Cycle Proteins
  • Chromatin
  • Crb2 protein, S pombe
  • Histones
  • Luminescent Proteins
  • Nuclear Proteins
  • Phosphoproteins
  • Schizosaccharomyces pombe Proteins
  • Serine
  • Lysine