STRUCTURE OF CHIMERIC DUPLEX JUNCTIONS - SOLUTION CONFORMATION OF THERETROVIRAL OKAZAKI-LIKE FRAGMENT R(CCCA)D(AATGA)CENTER-DOT-D(TCATTTGGG) FROM MOLONEY MURINE LEUKEMIA-VIRUS

We have determined the solution structure of the synthetic chimeric du plex r(ccca)(AATGA). d(TCATTTGGG) by two-dimensional NMR, distance geo metry, restrained molecular dynamics, and full relaxation matrix simul ation of the two-dimensional nuclear Overhauser effect spectra at vari ous mixing times. The chimeric strand of this duplex consists of the l ast four residues of the tRNA(Pro) primer for (-) strand DNA synthesis of Moloney murine leukemia virus and the first five residues of the ( -) strand DNA produced by extending this primer; the complementary DNA strand corresponds to the (+) strand product from this template. The hybrid section of this chimeric duplex assumes a structure similar to that found for pure hybrid duplexes of mixed sequence, while the DNA s ection assumes a conformation closer to B-form DNA. There is significa nt distortion of the duplex at the hybrid-DNA junction which is manife sted in marked changes in the helical parameters buckle, roll, and tip , changes in glycosidic torsion angles, and changes in the backbone to rsion angles delta, epsilon, and zeta. The sugar conformations also un dergo large changes, from heteromerous puckers in the hybrid section t o a more B-form in the DNA section. Furthermore, the intrastrand phosp hate separation in the chimeric strand is more typical of A-form duple xes in the RNA section but more like B-form duplexes in the DNA sectio n. In the DNA section the minor ve width changes gradually from B-form at the periphery and approaches hybrid-like dimensions closer to the junction. The structural discontinuities act synergistically to produc e a bend of 18 +/- 3 degrees at the junction. The global structure of this sequence is similar to that previously found in the chemically an alogous Okazaki Fragment r(gcg)d(TATACCC). d(GGGTATACGC) in solution. Such structure homology suggests a possible link between structure and function with respect to the recognition and cleavage of the junction RNA residues in both retroviral chimeras and Okazaki fragments during reverse transcription and normal DNA replication.