Structure of a DNA : RNA Hybrid Duplex: Why RNase H Does Not Cleave Pure RNA

Abstract

The solution structure of the DNA : RNA hybrid duplex d(GTCACATG) : r(caugugac) has been determined by means of two-dimensional nuclear Overhauser effect (2D-NOE) spectra, restrained molecular dynamics and full-relaxation matrix stimulation of the 2D-NOE spectra. The DNA : RNA hybrid duplex assumes neither an A-form nor a B-form structure in solution, but an intermediate heteromerous duplex structure. The sugars of the RNA strand have a normal N-type C3′- endo conformation, but the DNA strand sugars have neither N-type nor S-type conformations; instead, they have an unexpected intermediate O4′- endo conformation. The negative x -displacement, as well as the small rise and positive inclination of the base-pairs, resembles A-form morphology but the minor groove width is intermediate between that of A-form and B-form duplexes. Both the DNA and RNA strands show prominent sequence-dependent variations in their helical parameters. Combined analysis of NOE and J-coupling data indicates that the DNA sugars are not in a dynamical two-state equilibrium. The detailed three-dimensional structure of this DNA : RNA hybrid molecule leads to a proposed model for its interaction with RNase H. Several specific structural features of the enzyme complexed with the hybrid duplex appear to explain the mechanism whereby RNase H discriminates between DNA : RNA hybrid duplexes and pure RNA : RNA duplexes.