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Newly-Identified Fragile Sites Push Stressed Cells toward Cancer

Replication-induced DNA damage is a potent source of genome instability, but the affected sites are largely uncharacterized. Using high throughput assays, Barlow, Faryabi, et. al., generated a genome-wide map of early replication-induced DNA damage sites (ERFS), which are distinct from well characterized common fragile sites (CFS). ERFS correlate with regions of high transcription and, as illustrated in the figure, can arise from the collision of the transcription and replication machinery.
Replication-induced DNA damage is a potent source of genome instability, but the affected sites are largely uncharacterized. Using high throughput assays, Barlow, Faryabi, et. al., generated a genome-wide map of early replication-induced DNA damage sites (ERFS), which are distinct from well characterized common fragile sites (CFS). ERFS correlate with regions of high transcription and, as illustrated in the figure, can arise from the collision of the transcription and replication machinery.

The genomes of cancer cells are often riddled with chromosomal alterations such as amplifications, deletions, and even translocations, where a piece of one chromosome breaks off and attaches to another. Understanding how these changes arise can give researchers new insights into the process of cancer development.

In a recent study, Andre Nussenzweig, Ph.D., in CCR’s Laboratory of Genome Integrity, and his colleagues investigated the regions of DNA susceptible to double-strand breaks in proliferating B cells. Because activated B cells rapidly divide while undergoing the programmed DNA rearrangements necessary to generate targeted antibodies, they are likely to be sensitive to stress during replication, the process of copying a cell’s genome before it divides.

Using mouse B cells as a model, the researchers first identified those sites along the B cell DNA that are likely to be damaged during replication stress. Treatment with hydroxyurea (HU), a drug that causes early replication stress by depleting the pool of nucleotides used to synthesize DNA, increased the amount of breakage-prone single-stranded DNA, which the scientists identified by its association with the protein RPA. The researchers reasoned that the segments of single-stranded DNA most sensitive to replication stress would co-localize with DNA repair proteins including BRCA1 and SMC5. Indeed, they found 2,204 regions where all three proteins associated with the DNA and called these early replicating fragile sites (ERFS).

The researchers noted that ERFS are enriched in repetitive sequences and in guanine-cytosine (GC) content. Previous studies had linked regions of elevated GC content to translocation break points in B cell lymphomas, supporting the idea that the ERFS may be areas susceptible to stress-induced damage. Most ERFS overlapped with gene coding sequences or their promoters and were transcribed more often than surrounding DNA. Because many of the ERFS were relatively close together (within 300,000 base pairs) and likely to be replicated together, the investigators grouped them into 619 hotspots. Among these, the investigators found 15 with the greatest RPA/BRCA1/SMC5 binding, and eight of these 15 regions are often rearranged in B cell lymphomas. Interestingly, none of the 619 ERFS hotspots overlap with eight common fragile sites (CFS), regions previously identified as sites of replication-induced DNA damage in cancer.

To determine whether chromosomal breaks actually occur at the ERFS, the researchers treated primary mouse B cells with HU and examined the structure of the metaphase chromosomes. They observed more chromosomal breaks in cells treated with HU than in untreated cells. Between eight and 15 percent of these breaks occurred at each of the six ERFS hotspots they examined. Conversely, none of the breaks corresponded with two of the most fragile CFS. When the scientists treated B cells with aphidicolin, which inhibits DNA polymerase, however, they found chromosomal breaks at the CFS but not the ERFS. These results suggest that ERFS are damaged when replication stalls at an early stage while CFS are broken later when replication fails. Thus, ERFS represent a new class of fragile site.

The researchers next wanted to understand how breaks at the ERFS hotspots might be regulated. Since the kinase ATR is known to play a role in protecting the genome from double-strand breaks, they treated B cells with an ATR inhibitor and looked for changes in chromosome structure. ATR inhibition resulted in breaks at both CFS and ERFS, suggesting the importance of ATR activity in preventing breaks at both types of fragile sites. Another protein, AID, causes programmed DNA damage in B cells that can lead to oncogene activation and double-strand breaks. The investigators treated normal B cells or B cells lacking AID with the ATR inhibitor and examined their chromosomal structures. Unlike the breaks at the IgH locus, a known target of AID, there was no difference in the number of breaks at the ERFS with or without AID, indicating that ERFS breaks arise independently of AID.

The scientists also investigated whether the level of transcription at an ERFS hotspot affected its sensitivity to damage. They determined the extent of chromosomal breaks in B and T cells treated with the ATR inhibitor at two ERFS, one with equal expression in both cell types and a second more highly expressed in B cells. The frequency of breaks at the first hotspot was similar in the two cell types. In contrast, the researchers observed about three times more breaks at the second hotspot in B cells relative to T cells in which the region was silent. These results suggest that specific ERFS hotspots have distinct sensitivities in different cell types depending on whether or not the region is transcribed. They also suggest the possibility that ERFS may arise as a result of collisions between replication and transcription machineries (see Figure 1).

Oncogene activation often leads to chromosomal alterations. To see whether an oncogene could trigger breaks at fragile sites, the investigators over-expressed c-myc in primary B cells. Compared to control cells, the c-myc over-expressing cells had almost double the amount of DNA damage, which was concentrated at several ERFS hotspots and one CFS. The researchers also found breaks at ERFS hotspots in a mouse model of replication stress and even observed a translocation between an ERFS and the IgH locus in one of these cells, mimicking a translocation found in human B cell lymphoma. Similarly, in biopsies of patients with diffuse large B cell lymphoma, regions of DNA amplifications and deletions overlapped with areas analogous to the mouse ERFS. In fact, 25 of 64 genes known to contribute to cancer formation in a number of tissues are located at ERFS, implicating these sites in cancers beyond B cell lymphomas.

Reference
Barlow JH, Faryabi RB, Callén E, Wong N, Malhowski A, Chen HT, Gutierrez-Cruz G, Sun HW, McKinnon P, Wright G, Casellas R, Robbiani DF, Staudt L, Fernandez-Capetillo O, and Nussenzweig A. Identification of Early Replicating Fragile Sites that Contribute to Genome Instability. Cell. January 24, 2013. Pubmed Link