Hairpin induced slippage and hyper-methylation of the fragile X DNA triplets

The fragile X triplet repeats, (GCC)(n) .(GGC)(n), are located at the 5' untranslated region of the FMR-1 gene. Inordinate repeat expansion and hyper-methylation of the CpG islands inside the repeat lead to the suppression of the FMR-1 gene and the subsequent onset and progression of the disease. Previously, we have shown that the (GCC)(n) strand of the fragile X repeat readily forms hairpin structures under physiological conditions (Chen et al., Proc. Natl. Acad. Sci. USA, 92:5199-5203, 1995; Mariappan et al., Nucl. Acid Res. 24:784-792, 1996). Here, we show by an in vitro assay that formation of the (GCC)n hairpins leads to slippage during replication. The slippage structure is a three-way junction with two Watson-Crick, (GCC)(n) .(GGC)(n), arms and a third (GCC)(n) hairpin arm. Formation of such slippage structures during replication may explain the observed length polymorphism of the fragile X repeat. We have also studied the substrate efficiency of these three-way junctions toward the human methyltransferase, the enzyme that methylates the CpG sites in DNA. These methylation studies show that the slippage structures induced by the (GCC)(n) hairpins are 10-15 times more efficient substrates than either the corresponding Watson-Crick duplexes or the (GCC)(n) hairpins. We demonstrate by appropriate designs that the exceptional substrate efficiency of the three-way junction slippage structures is due to two factors: (i) the presence of the (GCC)(n) hairpin in which CpG sites are more accessible for methylation than the CpG sites in the Watson-Crick duplex and (ii) the ability of the (GCC)(n) hairpin in these three-way junctions to move along the Watson-Crick arms that facilitates conversion of low-affinity Watson-Crick CpG sites into high-affinity hairpin CpG sites. Therefore, we suggest that the formation of the (GCC)(n) hairpins during replication can explain both length polymorphism and hyper-methylation of the fragile X repeats.