STRUCTURE AND DYNAMICS OF THE DNA HAIRPINS FORMED BY TANDEMLY REPEATED CTG TRIPLETS ASSOCIATED WITH MYOTONIC-DYSTROPHY

Anomalous expansion of the DNA triplet (CTG)n causes myotonic dystroph y. Structural studies have been carried out on (CTG)n repeats in an at tempt to better understand the molecular mechanism of repeat expansion . NMR and gel electrophoretic studies demonstrate the presence of hair pin structures for (CTG)(5) and (CTG)(6) in solution. The monomeric ha irpin structure remains invariant over a wide range of salt concentrat ions (10-200 mM NaCl), DNA concentrations (micromolar to millimolar in DNA strand) and pH (6.0-7.5). The (CTG) hairpin contains three bases in the loop when n is odd and four bases when n is even. For both odd and even n the stacking and pairing in the stem remain the same, i.e. two hydrogen bond T . T pairs stack with the neighboring G C pairs. Al l the nucleotides in (CTG)5 and (CTG)(6) adopt C2'-endo, anti conforma tions. Full-relaxation matrix analysis has been performed to derive th e NOE distance constraints from NOESY experiments at seven different m ixing times (25, 50, 75, 100, 125, 200 and 500 ms). NOESY-derived dist ance constraints were subsequently used in restrained molecular dynami cs simulations to obtain a family of structures consistent with the NM R data. The theoretical order parameters are computed for H5-H6 (cytos ines) and H2'-H2'' dipolar correlations for both (CTG)5 and (CTG)(6) b y employing the Lipari-Szabo formalism. Experimental data show that th e cytosine in the loop of the (CTG)(5) hairpin is slightly more flexib le than those in the stem. The cytosine in the loop of the (CTG)(6) ha irpin is extremely flexible, implying that the dynamics of the four ba se loop is intrinsically different from that of the three base loop.