Hairpin Induced Slippage and Hyper-methylation of the Fragile X DNA Triplets

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Abstract
The fragile X triplet repeats, (GCC)n•(GGC)n, are located at the 5′ untranslated region of the FMRA 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)., 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)., 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)., 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.