119 Fork stalling at AT-rich sequences and failure of origin activation lead to chromosomal instability at fragile sites

Abstract

Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Using DNA combing, we studied the replication dynamics along two common fragile sites on chromosome 16, FRA16C and FRA16D. We found in FRA16C that under normal growth conditions, the replication along the fragile region shows stress-like dynamics. Replication forks along the fragile site progress significantly slower than in the entire genome, and frequently stall at AT-rich sequences. Interestingly, under these conditions, most of the stalled forks are concentrated near the largest 3.3Kbs AT-rich sequence. Furthermore, the distance between origins along FRA16C are significantly shorter than in the entire genome. Together, these results indicate that even under normal growth conditions, replication along the fragile site is continuously perturbed leading to activation of additional origins to enable replication of the region. Under mild replication stress induced by aphidicolin, the replication rate at the FRA16C region is further slowed down and the frequency of fork stalling at AT-rich sequences is further increased. Strikingly, unlike the entire genome, additional origins are not activated suggesting that all potential origins in the FRA16C region are already activated under normal conditions. These results demonstrate the inability of FRA16C to compensate for replication stress leading to failure of normal replication completion (Ozeri-Galai et al., 2011). To analyse directly, the role of AT-rich sequences in fragile site expression we targeted the 3.3 Kbs long AT-rich sequence derived from FRA16C into a non-fragile genomic region using a homologous recombination-based system. The results of these experiments will be discussed. Replication dynamic analysis of another fragile site, FRA16D, which is enriched with AT-rich sequences, reveals for the first time that a combination of DNA sequences and abnormal origin activation underlies the molecular basis of replication stress sensitivity of a fragile site. Moreover, the results provide a novel mechanism underlying the replication delay and replication stress sensitivity of fragile sites and shed light on the basis of genomic instability during the early stages of cancer development.