BRCT domain interactions with phospho-histone H2A target Crb2 to chromatin at double-strand breaks and maintain the DNA damage checkpoint.
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.
Sofueva
Sevil
S
Department of Molecular Biology, MB3, the Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Du
Li-Lin
LL
Limbo
Oliver
O
Williams
Jessica S
JS
Russell
Paul
P
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D002843:Chromatin; C109867:Crb2 protein, S pombe; D006657:Histones; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D029702:Schizosaccharomyces pombe Proteins; D012694:Serine; D008239:Lysine