Cell Cycle-dependent Complex Formation of BRCA1·CtIP·MRN Is Important for DNA Double-strand Break Repair

Abstract

BRCA1 plays an important role in the homologous recombination (HR)-mediated DNA double-strand break (DSB) repair, but the mechanism is not clear. Here we describe that BRCA1 forms a complex with CtIP and MRN (Mre11/Rad50/Nbs1) in a cell cycle-dependent manner. Significantly, the complex formation, especially the ionizing radiation-enhanced association of BRCA1 with MRN, requires cyclin-dependent kinase activity. CtIP directly interacts with Nbs1. The in vivo association of BRCA1 with MRN is largely dependent on the association of CtIP with the BRCT domains at the C terminus of BRCA1, whereas the N terminus of BRCA1 also contributes to its association with MRN. CtIP, as well as the interaction of BRCA1 with CtIP and MRN, is critical for IR-induced single-stranded DNA formation and cellular resistance to radiation. Consistently, CtIP itself is required for efficient HR-mediated DSB repair, like BRCA1 and MRN. These studies suggest that the complex formation of BRCA1⅐CtIP⅐MRN is important for facilitating DSB resection to generate single-stranded DNA that is needed for HR-mediated DSB repair. Because cyclin-dependent kinase is important for establishing IR-enhanced interaction of MRN with BRCA1, we propose that the cell cycle-dependent complex formation of BRCA1, CtIP, and MRN contributes to the activation of HR-mediated DSB repair in the S and G 2 phases of the cell cycle.

CtIP-mediated resection is essential for viability and can operate independently of BRCA1

“…To circumvent this, we generated a B cell-specific deletion of CtIP by intercrossing CD19 cre mice (Rickert et al, 1997) with mice carrying conditional and null alleles (CtIP co/2 ; Chen et al, 2008;Bothmer et al, 2013). The resulting CtIP-null (CtIP /2 ) B cells expressed little or no CtIP protein as determined by Western blot analysis ( Fig.…”

“…Tumor suppressor functions of BRCA1 are mediated by the BRCA1 carboxyl-terminal (BRCT) domain (Shakya et al, 2011), a motif that binds phosphorylated serine motifs in three different DNA repair proteins: BACH1 (BRIP1), ABRAXAS (CCDC98), and CtIP (Rbbp8; Li and Greenberg, 2012). Among these complexes, BRCA1 association with CtIP has been implicated in nucleolytic resection of DNA double strand breaks (DSBs; Sartori et al, 2007;Chen et al, 2008). damage signaling, genome instability, and defective resection.…”

BRCA1, PARP, and 53BP1: conditional synthetic lethality and synthetic viability

“…BRCA1 is also recruited to stalled replication forks during S-phase, and interacts with CtIP and the MRE11/Rad50/NBS complex to regulate end resection activity (Scully et al, 1997a;Yu et al, 1998;Sartori et al, 2007;Chen et al, 2008). These data taken together suggest there may be a functional interaction between BRCA1 and PARP1/2 in regulating DNA end-processing activity at stalled replication forks.…”

“…The interaction of BRCA1 with CtIP maybe especially critical for the regulation of processing of DNA ends at DSBs to promote HR (Sartori et al, 2007;Chen et al, 2008). BRCA1 also interacts with PALB2 linking it functionally to BRCA2 and its role in RAD51 loading during HR (Xia et al, 2006;Sy et al, 2009;Zhang et al, 2009a,b).…”

“…Thus, the BRCT domains of BRCA1 likely mediate critical interactions required for assembly of protein complexes required for proper DNA repair function and tumor suppression. This includes interaction of BRCA1 BRCT domains with: (i) BACH1/BRIP1 and TOBP1 (Cantor et al, 2001;Greenberg et al, 2006); (ii) Abraxas and RAP80 (Kim et al, 2007;Liu et al, 2007b;Wang et al, 2007); and (iii) CtIP and the MRE11/NBS/RAD50 complex (Wong et al, 1998;Sartori et al, 2007;Chen et al, 2008).…”