DNA instability at chromosomal fragile sites in cancer

Abstract

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.

Keywords: ATR checkpoint pathway; DNA secondary structure; RET/PTC rearrangement; cancer-specific chromosomal translocation; environmental mutagen; fragile site; stalled replication fork..

Fig. (1)

Model of fragile site instability in the formation of cancer-specific chromosomal rearrangements. RET and CCDC6 genes, located on chromosome 10 within the fragile sites FRA10G and FRA10C respectively, are closer than expected during interphase in normal thyroid cells. In the normal thyroid gland, RET is not expressed in follicular cells, while CCDC6 is constitutively expressed. Under conditions of replication stress, replicative DNA polymerases α, δ, and ε become uncoupled from the helicase/topoisomerase complex, resulting in long stretches on single-stranded DNA susceptible to the formation of stable secondary structures. These structures can cause replication fork stalling, triggering the ATR-dependent DNA damage checkpoint pathway. Fragile sites may also be susceptible to spontaneous fork reversal or polymerase skipping at regions of secondary structure. For repair of stalled forks, ATR binds to the fragile DNA either directly or through complexes, and activates a downstream signaling cascade with other proteins, including CHK1, BRCA1, FANCD2, WRN, Claspin, HUS1, ATM, and SMC1. If the ATR pathway properly responds, the replication fork will be repaired and DNA replication will resume normally. A loss, deficiency, or defect in ATR pathway proteins could lead to checkpoint failure and/or replication fork collapse resulting in DNA breakage at RET and CCDC6. DNA breakage at these sites can lead to the formation of RET/PTC1 translocations, the expression of oncogenic RET protein, and the development of papillary thyroid carcinoma.