Structural and functional analysis of the Crb2–BRCT₂ domain reveals distinct roles in checkpoint signaling and DNA damage repair

Abstract

Schizosaccharomyces pombe Crb2 is a checkpoint mediator required for the cellular response to DNA damage. Like human 53BP1 and Saccharomyces cerevisiae Rad9 it contains Tudor₂ and BRCT₂ domains. Crb2-Tudor₂ domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT₂ domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2–BRCT₂ domain, alone and in complex with a phosphorylated H2A.1 peptide, reveals the structural basis for dimerization and direct interaction with γ-H2A.1 in ionizing radiation-induced foci (IRIF). Mutational analysis in vitro confirms the functional role of key residues and allows the generation of mutants in which dimerization and phosphopeptide binding are separately disrupted. Phenotypic analysis of these in vivo reveals distinct roles in the DNA damage response. Dimerization mutants are genotoxin sensitive and defective in checkpoint signaling, Chk1 phosphorylation, and Crb2 IRIF formation, while phosphopeptide-binding mutants are only slightly sensitive to IR, have extended checkpoint delays, phosphorylate Chk1, and form Crb2 IRIF. However, disrupting phosphopeptide binding slows formation of ssDNA-binding protein (Rpa1/Rad11) foci and reduces levels of Rad22(Rad52) recombination foci, indicating a DNA repair defect.

Keywords

• Checkpoint mediator
• crystal structure
• histone code
• phosphopeptide binding