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

Kilkenney, M L, Dore, A, Roe, S M, Nestoras, K, Ho, J, Watts, F Z and Pearl, L (2008) Structural and functional analysis of the Crb2-BRCT2 domain reveals distinct roles in checkpoint signaling and DNA damage repair. Genes and Development, 22 (15). pp. 2034-2047. ISSN 0890-9369

[img] PDF
Restricted to SRO admin only

Download (1MB)

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 Tudor(2) and BRCT(2) domains. Crb2-Tudor(2) domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT(2) domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2-BRCT(2) domain, alone and in complex with a phosphorylated H2A.1 peptide, reveals the structural basis for dimerization and direct interaction with gamma-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.

Item Type: Article
Additional Information: GDSC261
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Depositing User: Gee Wheatley
Date Deposited: 28 Oct 2008
Last Modified: 07 Mar 2017 04:19
URI: http://sro.sussex.ac.uk/id/eprint/2057

View download statistics for this item

📧 Request an update