Replication Delay along FRA7H, a Common Fragile Site on Human Chromosome 7, Leads to Chromosomal Instability

Fragile sites are specific chromosomal loci prone to breakage, characterized by constrictions, gaps, or breaks on chromosomes from cells exposed to specific tissue culture and chemical conditions (reviewed in reference 40). They are classified as either rare or common, depending on their frequency within the population and their mode of induction. Rare fragile sites (n = 30 in the human genome) appear in less than 5% of the human population and segregate in specific families. Common fragile sites (n = 90), on the other hand, are considered to be part of the normal chromosomal structure and are thought to present in all individuals. Most of the common fragile sites (n = 76) are induced by aphidicolin (7), an inhibitor of DNA polymerases alpha and delta (reviewed in reference 41). Several rare fragile sites, induced by folic acid depravation, dystamycin A, or bromodeoxyuridine (BrdU) have been characterized at the molecular level (16, 19, 20, 29, 31, 48). The expression of these sites is associated with expanded CGG trinucleotide or AT-rich minisatellite repeats. Three common fragile sites (FRA3B, FRA7G, and FRA7H), all induced by aphidicolin, were identified, cloned, and sequenced (15, 28, 46). The cytogenetic expression of each of these sites appears along a region of several hundred kilobases. No expanded repeats were found in these regions. Fragile sites were implicated in chromosomal rearrangement (8), gene amplification (4), sister chromatid exchange (9), and integration of foreign DNA (28, 32, 33, 47). This genetic instability can lead to disease manifestation (6, 16, 30) and might play a role in oncogenesis (49). Despite their instability, several common fragile sites are conserved between mouse and human (5, 8), indicating that these sites might play an important biological role. The molecular mechanism underlying the genetic instability at fragile sites is currently not understood. The fragility inducers interfere with DNA replication, and their effect is restricted to S phase (40). Replication inhibition of Drosophila cells resulted in a morphological appearance resembling the mammalian fragile sites at the intercalary heterochromatin regions known to replicate late in S phase. All of these findings led Laird et al. to suggest that fragile sites replicate very late in the cell cycle. Upon replication stress or premature chromatin condensation, the condensation of these sequences might not be completed, and a fragile site will appear (22). Analysis of the replication time of two rare fragile sites, FRAXA and FRAXE, showed that the normal alleles replicated very late in S phase (at S/G2), and the CGG-expanded alleles replicated even later, at G2 phase (11, 12, 39). These CGG-repeated sequences can adopt non-B-DNA structures that inhibit replication fork movement, both in vitro and in vivo (35, 43). The common fragile site FRA3B, or at least those FRA3B alleles that express fragility (45), has also been shown to replicate at the latest part of S phase (23). Following aphidicolin induction, ∼15% of the FRA3B alleles were unreplicated in the G2 phase (23). These findings support the model suggested by Laird et al. (21) and indicate that late replication may be a common feature of rare and common fragile sites (41). However, the basis for the replication delay in common fragile sites upon stress is unknown. Here, we analyze the replication pattern of a common fragile site, FRA7H, on the long arm of human chromosome 7. Our results suggest that perturbed fork progression, which results in delayed replication along the FRA7H region, underlines the fragility of this site.