SOLUTION STRUCTURES OF THE HUNTINGTONS-DISEASE DNA TRIPLETS, (CAG)(N)

Highly polymorphic DNA triplet repeats, (CAG)(n), are located inside t he first exon of the Huntington's disease gene. Inordinate expansion o f this repeat is correlated with the onset and progression of the dise ase. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific P1 enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)(n) expansion. Nondenaturing gel electrophoresis and 1D H-1 NMR studies of (CAG)(5) and (CAG)(6) re veal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations ( i.e., 1.0-2.0 mM that is typically required for 2D NMR experiments) bo th (CAG)(5) and (CAG)(6) exist predominantly in mismatched duplex form s. Mismatched duplex structures of (CAG)(5) and (CAG)(6) are useful, b ecause they adequately model the stem of the biologically relevant hai rpins formed by (CAG)(n). We, therefore, performed detailed NMR spectr oscopic studies on the duplexes of (CAG)(5) and (CAG)(6). We also stud ied a model duplex, (CGCAGCG)(2) that contains the underlined building block of the duplex. This duplex shows the following structural chara cteristics: (i) all the nucleotides are in (C2'-endo, anti) conformati ons, (ii) mismatched A.A base pairs are flanked by two Watson-Crick G. C base pairs and (iii) A.A base pairs are stably stacked (and intra-he lical) and are formed by a single N6-H-N1 hydrogen bond. The nature of A.A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CAG(5)](2) duplex where the adenines are N-15-l abeled at N6. Temperature-and pH-dependent imino proton spectra, nonde naturing electrophoresis, and P1 digestion data demonstrate that under a wide range of solution conditions longer (CAG)(n) repeats (n greate r than or equal to 10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)(8,21) inserts in the M13 single-stranded DNA templates shows a replication bypass for the (CAG)(21) insert but not for the (CAG)(8) i nsert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)(n) even i n presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)(n) may cause slippage during rep lication and thus may explain the observed length polymorphism.