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Okazaki fragments are important intermediates in DNA replication. Chimeric duplexes that are structurally equivalent to Okazaki fragments also occur during reverse transcription of RNA retroviruses. Such duplexes consist of an RNA-DNA chimeric strand base-paired to a pure DNA strand; hence they have a hybrid duplex "left half" covalently linked to a "right half" that is pure DNA. We have determined the solution structure of the synthetic Okazaki fragment r(gcg)d(TATACCC):d(GGGTATACGC) by means of two-dimensional NMR, restrained molecular dynamics and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The large negative x-displacement and large positive inclination in the hybrid section of the duplex are structural characteristics similar to those found in pure hybrid 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 assumes a conformation in solution that is a chimeric mixture of hybrid-form (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 approximately 18.1 (+/- 0.4) degrees at the junction between the hybrid and DNA segments that may be important for reverse transcriptase binding and RNase H cleavage of such molecules. Despite the fact that the solution structure is quite different from the all A-form structure reported recently for the exact same molecule in the crystalline state, a surprising number of local helical parameters were found to be quite similar to those reported for the crystal structure.

Type

Journal article

Journal

J Mol Biol

Publication Date

19/08/1994

Volume

241

Pages

440 - 455

Keywords

Base Sequence, Computer Graphics, DNA, DNA Replication, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Molecular Structure, Nucleic Acid Conformation, Oligodeoxyribonucleotides, Oligoribonucleotides