Replication stress induces genome-wide copy number changes in human cells that resemble polymorphic and pathogenic variants.

Copy number variants (CNVs) are an important component of genomic variation in humans and other mammals. Similar de novo deletions and duplications, or copy number changes (CNCs), are now known to be a major cause of genetic and developmental disorders and to arise somatically in many cancers. A major mechanism leading to both CNVs and disease-associated CNCs is meiotic unequal crossing over, or nonallelic homologous recombination (NAHR), mediated by flanking repeated sequences or segmental duplications. Others appear to involve nonhomologous end joining (NHEJ) or aberrant replication suggesting a mitotic cell origin. Here we show that aphidicolin-induced replication stress in normal human cells leads to a high frequency of CNCs of tens to thousands of kilobases across the human genome that closely resemble CNVs and disease-associated CNCs. Most deletion and duplication breakpoint junctions were characterized by short (<6 bp) microhomologies, consistent with the hypothesis that these rearrangements were formed by NHEJ or a replication-coupled process, such as template switching. This is a previously unrecognized consequence of replication stress and suggests that replication fork stalling and subsequent error-prone repair are important mechanisms in the formation of CNVs and pathogenic CNCs in humans.

Replication stress induces tumor-like microdeletions in FHIT/FRA3B.

Common fragile sites (CFSs) are loci that preferentially exhibit metaphase chromosome gaps and breaks after partial inhibition of DNA synthesis. The fragile site FRA3B, which lies within the FHIT tumor-suppressor gene, is a site of frequent heterozygous and homozygous deletions in many cancer cells and precancerous lesions. The great majority of FHIT and other CFS-associated gene rearrangements in tumors are submicroscopic, intralocus deletions of hundreds of kilobases that often result in inactivation of associated genes. Although CFS instability leads to chromosome gaps and breaks and translocations, there has been no direct evidence showing that CFS instability or replication stress can generate large submicroscopic deletions of the type seen in cancer cells. Here, we have produced FHIT/FRA3B deletions closely resembling those in tumors by exposing human-mouse chromosome 3 somatic hybrid cells to aphidicolin-mediated replication stress. Clonal cell populations were analyzed for deletions by using PCR, array comparative genomic hybridization (aCGH), and FISH. Thirteen percent to 23% of clones exhibited submicroscopic FHIT deletions spanning approximately 200-600 kb within FRA3B. Chromosomes with FRA3B deletions exhibited significantly decreased fragility of this locus, with a 2- to 12-fold reduction in metaphase gaps and breaks compared with controls. Sequence analysis showed no regions of homology at breakpoints and suggests involvement of NHEJ in generating the deletions. Our results demonstrate that replication stress induces a remarkably high frequency of tumor-like microdeletions that reduce fragility at a CFS in cultured cells and suggests that similar conditions during tumor formation lead to intralocus deletion and inactivation of genes at CFSs and perhaps elsewhere in the genome.

Chromosome fragile sites.

Chromosomal fragile sites are specific loci that preferentially exhibit gaps and breaks on metaphase chromosomes following partial inhibition of DNA synthesis. Their discovery has led to novel findings spanning a number of areas of genetics. Rare fragile sites are seen in a small proportion of individuals and are inherited in a Mendelian manner. Some, such as FRAXA in the FMR1 gene, are associated with human genetic disorders, and their study led to the identification of nucleotide-repeat expansion as a frequent mutational mechanism in humans. In contrast, common fragile sites are present in all individuals and represent the largest class of fragile sites. Long considered an intriguing component of chromosome structure, common fragile sites have taken on novel significance as regions of the genome that are particularly sensitive to replication stress and that are frequently rearranged in tumor cells. In recent years, much progress has been made toward understanding the genomic features of common fragile sites and the cellular processes that monitor and influence their stability. Their study has merged with that of cell cycle checkpoints and DNA repair, and common fragile sites have provided insight into understanding the consequences of replication stress on DNA damage and genome instability in cancer cells.

Common fragile sites as targets for chromosome rearrangements.

Common fragile sites are large chromosomal regions that preferentially exhibit gaps or breaks after DNA synthesis is partially perturbed. Fragile site instability in cultured cells is well documented and includes gaps and breaks on metaphase chromosomes, translocation and deletions breakpoints, and sister chromosome exchanges. In recent years, much has been learned about the genomic structure at fragile sites and the cellular mechanisms that monitor their stability. The study of fragile sites has merged with that of cell cycle checkpoints and DNA repair, with multiple proteins from these pathways implicated in fragile site stability, including ATR, BRCA1, CHK1, and RAD51. Since their discovery, fragile sites have been implicated in constitutional and cancer chromosome rearrangements in vivo and recent studies suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early tumorigenesis. Here we review the relationship of fragile sites to chromosome rearrangements, particularly in tumor cells, and discuss the mechanisms that may be involved.

Mechanisms of common fragile site instability.

The study of common fragile sites has its roots in the early cytogenetic investigations of the fragile X syndrome. Long considered an interesting component of chromosome structure, common fragile sites have taken on novel significance as regions of the genome that are particularly sensitive to certain forms of replication stress, which are frequently rearranged in cancer cells. In recent years, much has been learned about the genomic structure at fragile sites and the cellular checkpoint functions that monitor their stability. Recent findings suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early stages of tumorigenesis. Thus, the study of common fragile sites can provide insight not only into the nature of fragile sites, but also into the broader consequences of replication stress on DNA damage and cancer. However, despite recent advances, many questions remain regarding the normal functional significance of these conserved regions and the basis of their fragility.

The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability.

Fanconi anemia (FA) is a rare multi-genic, autosomal and X-linked recessive disorder characterized by hematological abnormalities, developmental defects and increased cancer susceptibility. Patient-derived FA cells display heightened sensitivity to DNA cross-linking agents such as mitomycin C (MMC). In response to DNA damaging agents, and during S-phase of the cell cycle, the FA pathway is activated via the mono-ubiquitination of FANCD2 (FANCD2-Ub), signaling its translocation to discrete nuclear foci, where it co-localizes with the central DNA repair proteins BRCA1 and RAD51. However, the exact function of activated FANCD2-Ub remains unclear. Here, we have characterized the role of the FA pathway in response to DNA replicative stress by aphidicolin (APH) and hydroxyurea (HU). The FA pathway is strongly activated in response to both agents. In addition, using patient-derived FA cell lines and siRNA targeting FANCD2, we demonstrate a functional requirement for the FA pathway in response to low doses of APH: a replicative stress treatment known to result in chromosome breakage at common fragile sites. Both the total number of chromosome gaps and breaks and breaks at the specific common fragile sites FRA3B and FRA16D were significantly elevated in the absence of an intact FA pathway. Furthermore, we demonstrate that APH activates the mono-ubiquitination of both FANCD2 and PCNA and the phosphorylation of RPA2, signaling processive DNA replication arrest. Following APH treatment, FANCD2-Ub co-localizes with PCNA (early) and RPA2 (late) in discrete nuclear foci. Our results demonstrate an integral role for the FA pathway in the DNA replication stress response.

Chromosomal instability at common fragile sites in Seckel syndrome.

Seckel syndrome (SCKL) is a rare, genetically heterogeneous disorder, with dysmorphic facial appearance, growth retardation, microcephaly, mental retardation, variable chromosomal instability, and hematological disorders. To date, three loci have been linked to this syndrome, and recently, the gene encoding ataxia-telangiectasia and Rad3-related protein (ATR) was identified as the gene mutated at the SCKL1 locus. The ATR mutation affects splicing efficiency, resulting in low levels of ATR in affected individuals. Elsewhere, we reported increased instability at common chromosomal fragile sites in cells lacking the replication checkpoint gene ATR. Here, we tested whether cells from patients carrying the SCKL1 mutation would show increased chromosome breakage following replication stress. We found that, compared with controls, there is greater chromosomal instability, particularly at fragile sites, in SCKL1-affected patient cells after treatment with aphidicolin, an inhibitor of DNA polymerase alpha and other polymerases. The difference in chromosomal instability between control and patient cells increases at higher levels of aphidicolin treatment, suggesting that the low level of ATR present in these patients is not sufficient to respond appropriately to replication stress. This is the first human genetic syndrome associated with increased chromosome instability at fragile sites following replication stress, and these findings may be related to the phenotypic findings in patients with SCKL1.

BRCA1 is required for common-fragile-site stability via its G2/M checkpoint function.

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 aphidicolin-induced 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.

Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by features reminiscent of marked premature ageing. Here, we present evidence of mutations in lamin A (LMNA) as the cause of this disorder. The HGPS gene was initially localized to chromosome 1q by observing two cases of uniparental isodisomy of 1q-the inheritance of both copies of this material from one parent-and one case with a 6-megabase paternal interstitial deletion. Sequencing of LMNA, located in this interval and previously implicated in several other heritable disorders, revealed that 18 out of 20 classical cases of HGPS harboured an identical de novo (that is, newly arisen and not inherited) single-base substitution, G608G(GGC > GGT), within exon 11. One additional case was identified with a different substitution within the same codon. Both of these mutations result in activation of a cryptic splice site within exon 11, resulting in production of a protein product that deletes 50 amino acids near the carboxy terminus. Immunofluorescence of HGPS fibroblasts with antibodies directed against lamin A revealed that many cells show visible abnormalities of the nuclear membrane. The discovery of the molecular basis of this disease may shed light on the general phenomenon of human ageing.