Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability. The disorder is named for a fragile site on the X chromosome that is seen when patient cells are treated with agents like folate or 5-fluorodeoxyuridine (FdU) that affect a key enzyme in the pyrimidine biosynthetic pathway, thymidylate synthase (1
). This causes a nucleotide pool imbalance thereby slowing replication and perhaps requiring repair to remove misincorporated bases. The fragile site appears as a gap, constriction or break in metaphase chromosomes. The Fragile X fragile site is also known as FRAXA (Fra
gile site on X
chromosome, site A
). FRAXA is coincident with a stretch of >200 CGG•CCG-repeats in the 5′ UTR of the FMR1
gene that is responsible for FXS (3
). Alleles with this number of repeats arise from an increase in the number of repeats on maternal transmission of an allele with 55–200 repeats. The relationship between this repeat expansion and chromosome fragility is unknown.
The sequence basis of six other folate-sensitive fragile sites in the human genome, FRAXE, FRAXF, FRA10A, FRA11A, FRA11B, FRA12A and FRA16A, have so far been determined. In all instances, the responsible sequence is also a long CGG•CCG-repeat tract (5–10
). FRAXA is a frequent translocation breakpoint in rodent-human somatic hybrids (4
). FRA11B, is associated with deletion of the telomeric end of long arm of chromosome 11 in a number of cases of Jacobsen Syndrome (12
More than 110 other fragile sites are found in human genomes, many of which are common translocation breakpoints associated with different forms of cancer. Many of these sites are induced by aphidicolin (APC) and 13 such sites have been studied so far at the sequence level. They are all associated with hundreds of kilobases or even megabases of DNA with no particularly distinctive sequences (5
). In the case of FRA3B, the most active APC-sensitive fragile site, breakage is seen in multiple places throughout a 4-Mb region and it has been suggested that no single sequence is responsible (28
). Agents that induce chromosome fragility all share with folate-stress the potential to interfere with DNA replication. APC, for example, is an inhibitor of DNA polymerase α, δ and ε (29
). At high concentrations these compounds may also affect DNA repair.
ATR and ATM are kinases responsible for the initiation and management of the DNA damage response (DDR) in mammals (31
). Depending on the type of DNA damage, one or both of these enzymes are activated. The activated protein then initiates a kinase cascade that activates key downstream effectors of the DDR. The ATR-pathway is thought to be involved primarily in the cellular response to stalled replication forks and bulky DNA lesions that block DNA synthesis, while the ATM pathway is thought to respond primarily to double-strand breaks although some cross-talk between these pathways is apparent. Both ATR and ATM have been shown to protect the genome against APC-sensitive chromosome fragility (19
Here we describe our analysis of FRAXA. Our data show that, as with the APC-sensitive sites, the ATR-pathway is involved in preventing FdU-sensitive chromosome fragility at FRAXA. KU55933, an inhibitor specific for ATM, reduces FdU-induced chromosome fragility. This suggests that a KU55933-sensitive enzyme, most likely ATM, is actually responsible for the generation of the fragile site. In addition, we show here that the Fragile X alleles exhibit a second form of fragility that is FdU-independent and that ATM is most likely responsible for preventing this form of chromosome fragility. This suggests that FRAXA differs in important ways from the APC-sensitive fragile sites and that FXS alleles form at least two different kinds of DNA lesions that gives rise to chromosome fragility, one that is normally repaired by ATR but not by ATM, and one that is repaired by ATM.