Published online 15 June 2004
Nucleic Acids Research, 2004, Vol. 32, No. 10 3228-3239
© 2004 Nucleic Acids Research, Vol. 32 No. 10 © Oxford University Press 2004; all rights reserved
Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold
Department of Physical Chemistry/Biophysical Chemistry, University of Nijmegen, Toernooiveld 1 6225 ED Nijmegen, The Netherlands, 1 Unilever Research and Development, Olivier van Noortlaan 120, 3133AT Vlaardingen, The Netherlands and 2 Department of Medical Biochemistry and Biophysics, Umeå University, S-90187 Umeå, Sweden
* To whom correspondence should be addressed. Tel: +031 24 3652678/3384; Fax: +031 24 3652112; Email: sybren.wijmenga{at}sci.kun.nl
Received April 12, 2004; Revised and Accepted May 19, 2004
Three-way junctions (3H) are the simplest and most commonly occurring branched nucleic acids. They consist of three double helical arms (A to C), connected at the junction point, with or without a number of unpaired bases in one or more of the three different strands. Three-way junctions with two unpaired bases in one strand (3HS2) have a high tendency to adopt either of two alternative stacked conformations in which two of the three arms A, B and C are coaxially stacked, i.e. A/B-stacked or A/C-stacked. Empirical stacking rules, which successfully predict for DNA 3HS2 A/B-stacking preference from sequence, have been extended to A/C-stacked conformations. Three novel DNA 3HS2 sequences were designed to test the validity of these extended stacking rules and their conformational behavior was studied by solution NMR. All three show the predicted A/C-stacking preference even in the absence of multivalent cations. The stacking preference for both classes of DNA 3HS2 can thus be predicted from sequence. The high-resolution NMR solution structure for one of the stacked 3HS2 is also reported. It shows a well-defined local and global structure defined by an extensive set of classical NMR restraints and residual dipolar couplings. Analysis of its global conformation and that of other representatives of the 3H family, shows that the relative orientations of the stacked and non-stacked arms, are restricted to narrow regions of conformational space, which can be understood from geometric considerations. Together, these findings open up the possibility of full prediction of 3HS2 conformation (stacking and global fold) directly from sequence.
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