BRCT Domain Interactions with Phospho-Histone H2A Target Crb2 to Chromatin at Double-Strand Breaks and Maintain the DNA Damage Checkpoint

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 (BRCT₂) 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 BRCT₂ 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 BRCT₂ 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.

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We thank Claire Dovey for assistance with microscopy, Steven Sanders for discussing data prior to publication, and members of the Russell laboratory and the Scripps Cell Cycle Groups for discussions.

S.S. is supported by a Skaggs-Oxford Scholarship, and L.-L.D. was a fellow of the Leukemia and Lymphoma Society. This work was funded by NIH grants GM59447 and CA77325 awarded to P.R. and CA117638 awarded to J. Tainer/P.R.