THE SOLUTION STRUCTURE OF THE R(GCG)D(TATACCC)-D(GGGTATACGC) OKAZAKI FRAGMENT CONTAINS 2 DISTINCT DUPLEX MORPHOLOGIES CONNECTED BY A JUNCTION

Okazaki fragments are important intermediates in DNA replication. Chim eric duplexes that are structurally equivalent to Okazaki fragments al so occur during reverse transcription of RNA retroviruses. Such duplex es consist of an RNA-DNA chimeric strand base-paired to a pure DNA str and; hence they have a hybrid duplex ''left half' covalently linked to a ''right hair' that is pure DNA. We have determined the solution str ucture of the synthetic Okazaki fragment r(gcg)d(TATACCC):d(GGGTATACGC ) by means of two-dimensional NMR, restrained molecular dynamics and f ull relaxation matrix simulation of the two-dimensional nuclear Overha user effect spectra at various mixing times. The large negative x-disp lacement and large positive inclination in the hybrid section of the d uplex are structural characteristics similar to those found in pure hy brid duplexes. However, the DNA sugar puckers and the width and depth of the minor groove in the pure DNA section are more like B-form DNA, especially beyond the junction. Thus, this Okazaki fragment duplex ass umes a conformation in solution that is a chimeric mixture of hybrid-f orm (H-form) and B-form structures and the overall molecule cannot be classified as either an A-form or a B-form duplex. The co-existence of these two different conformations in a single duplex gives rise to a structural discontinuity with a bend of similar to 18.1(+/-0.4)degrees at the junction between the hybrid and DNA segments that may be impor tant for reverse transcriptase binding and RNase H cleavage of such mo lecules. Despite the fact that the solution structure is quite differe nt from the all A-form structure reported recently for the exact same molecule in the crystalline state, a surprising number of local helica l parameters were found to be quite similar to those reported for the crystal structure.