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

J Biomol Struct Dyn. 1998 Feb;15(4):745-56. doi: 10.1080/07391102.1998.10508989.

Abstract

The fragile X triplet repeats, (GCC)n x (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 x (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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • DNA (Cytosine-5-)-Methyltransferases / metabolism
  • DNA / chemistry*
  • DNA Methylation*
  • DNA Replication
  • Fragile X Syndrome / genetics*
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Conformation*
  • Trinucleotide Repeats*

Substances

  • DNA
  • DNA (Cytosine-5-)-Methyltransferases