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

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

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  • 1Department of Molecular Biology, MB3, the Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.


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.

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