BRCA1 Is Required for Common-Fragile-Site Stability via Its G₂/M Checkpoint Function

Abstract

Common fragile sites are loci that form chromosome gaps or breaks when DNA synthesis is partially inhibited. Fragile sites are prone to deletions, translocations, and other rearrangements that can cause the inactivation of associated tumor suppressor genes in cancer cells. It was previously shown that ATR is critical to fragile-site stability and that ATR-deficient cells have greatly elevated fragile-site expression (A. M. Casper, P. Nghiem, M. F. Arlt, and T. W. Glover, Cell 111:779-789, 2002). Here we demonstrate that mouse and human cells deficient for BRCA1, due to mutation or knockdown by RNA interference, also have elevated fragile-site expression. We further show that BRCA1 functions in the induction of the G 2 /M checkpoint after aphidicolininduced replication stalling and that this checkpoint function is involved in fragile-site stability. These data indicate that BRCA1 is important in fragile-site stability and that fragile sites are recognized by the G 2 /M checkpoint pathway, in which BRCA1 plays a key role. Furthermore, they suggest that mutations in BRCA1 or interacting proteins could lead to rearrangements at fragile sites in cancer cells.Common fragile sites are loci that exhibit site-specific gaps and breaks on metaphase chromosomes when cells are grown under conditions that partially inhibit DNA synthesis, such as folate deficiency or treatment with aphidicolin (11). These fragile sites extend over hundreds of kilobases, with gaps and breaks occurring throughout the regions. Following aphidicolin treatment, 80% of all gaps and breaks are seen at just 20 fragile sites, with FRA3B (3p14.2) and FRA16D (16q23) being the most frequently broken, or expressed, fragile sites (11). Whereas rare fragile sites, such as FRAXA within the FMR1 gene, arise from mutation at di-or trinucleotide repeats, common fragile sites are found in all individuals and represent a normal component of chromosome structure.Fragile sites are so-called hot spots for sister chromatid exchanges, translocations, deletions, and plasmid integration in cultured cells following replication stress (12,14,32,40). Numerous studies have also shown that common fragile sites are prone to deletions and rearrangements in many cancers (1,17,21,28,33), and they may play a role in some gene amplification and viral integration events (6,16,26,41). Some fragile sites lie within putative tumor suppressor genes, such as FHIT at FRA3B and WWOX at FRA16D (30, 33), leading to the model that fragile-site instability is a contributing factor in tumorigenesis.Determining the mechanisms of fragile-site instability is important in understanding normal chromosome structure and DNA replication as well as the instability found at fragile sites in tumor cells. Sequence analysis of common fragile sites has not revealed why they are unstable. However, all sites studied to date are relatively AT rich and contain more areas of high flexibility than non-fragile-site regions (22,(24)(25)(26)(27). Studies examining replication timing at common fragile sites have shown...

Increased level of chromosomal damage after irradiation of lymphocytes from BRCA1 mutation carriers

“…Recently, it has been shown that BRCA1 is required for common fragile-site stability. Cells lacking BRCA1 show an increased expression of specific common fragile sites (Arlt et al, 2004). This provides further evidence that cells lacking BRCA1 are likely to be prone to genomic alterations that can lead to deletion of associated genes and this consequently could promote tumourigenesis.…”

The mINO80 chromatin remodeling complex is required for efficient telomere replication and maintenance of genome stability

“…This shift in tumor spectrum could be due to increased genomic instability stemming from telomere replication defects, an inability to engage in HDR-mediated repair, or alterations in gene transcription involved in tumorigenesis. Common fragile sites are vulnerable to DNA insertions and deletions, promoting an unstable genome that contributes to the formation of some human cancers [39,42]. We speculate that defects in telomere replication in the absence of mIno80 might lead to similar increases in genomic instability to promote tumorigenesis.…”

“…Telomeres resemble common fragile sites, difficult-to-replicate regions in the genome that, under conditions of replication stress, generate DSBs due to fork stalling [38][39][40][41]. Common fragile sites are vulnerable to DNA insertions and deletions, promoting genomic instability [39,42].…”

Understanding the molecular basis of common fragile sites instability: Role of the proteins involved in the recovery of stalled replication forks

“…45 Beside its role in sister chromatid cohesion, SMC1 is phosphorylated in an ATRdependent manner under conditions of replication stress and may contribute to the activation of the S-phase checkpoint after aphidicolin exposure, possibly allowing error-free resumption of DNA replication. 32,44 The observation that cells lacking BRCA1 show elevated levels of breakage at CFS after aphidicolin treatment, 33 represents, however, an interesting exception. Indeed, even though there is evidence that BRCA1 is phosphorylated by ATR in response to fork stalling, 46 its role in preventing breakage at CFS has been claimed not to be related to the S-phase checkpoint but rather to the G 2 /M checkpoint control regulating the onset of mitosis.…”

“…As a logical consequence of the previous findings and strengthening their consistency, other components of the ATR-dependent checkpoint were found to influence the stability of fragile sites. Among them are several ATR substrates or mediator proteins: CHK1, 4 HUS1, 30 Claspin, 31 SMC1 32 and BRCA1 33 ( Table 1).…”