Crystal Structure of an Eight-Base Pair Duplex Containing the 3‘-DNA−RNA-5‘ Junction Formed during Initiation of Minus-Strand Synthesis of HIV Replication

Abstract

During initiation of minus-strand synthesis by HIV-1 reverse transcriptase, a 3'-DNA-RNA-5' junction is formed involving the 3'-end of tRNAlys,3. The HIV-RT-associated RNase H cleaves the RNA template strand specifically, opposite the newly synthesized DNA strand. We have determined the crystal structure at 1.9 A resolution of an eight-base pair hybrid duplex representing the junction to identify global or local structural perturbations which may be recognized by HIV-RT RNase H. The junction octamer is in a global A-type conformation throughout. A base pair step with distinct stacking geometry and variable backbone conformation is located next to the main endonucleolytic cleavage site. This base pair step may serve as a recognition site for HIV-RT RNase H.

An Unusual Sugar Conformation in the Structure of an RNA/DNA Decamer of the Polypurine Tract May Affect Recognition by RNase H

“…14,15 This conformational backbone flexibility, primarily at one base removed in the 5 0 direction from the junction, has been proposed as a recognition signal for RNase H endolytic cleavage. 13 Szyperski et al 14 agree that the backbone conformations in this region are important. They make particular note of a sugar pucker variation at the hybrid junction and a surrounding base-step, where two of the DNA sugars interconvert between C3 0 -endo and C2 0 -endo.…”

“…However once (2 )-strand DNA synthesis begins, a chimeric hybrid, an Okazaki fragment, is formed at the junction of the growing DNA chain with the tRNA/RNA. This junction has been studied by both crystallography and NMR, 13,14 as has the Okazaki fragment generated at the completion of synthesis when the (2 )-strand primer is removed from the RNA/ DNA junction. 15 Another decamer RNA/DNA hybrid beginning one RNA nucleotide 5 0 of the cleavage site has been looked at crystallographically.…”

“…However, exceptions in sugar conformation at individual base-steps have been seen crystallographically, as well as other conformational variations along the backbones, such as abg crankshaft transitions. 13,16,26 The NMR solution studies note a difference in groove widths from standard A and B-form helices, with most hybrids having a minor groove width intermediate between A and B. The crystal structures, on the other hand, tend to have A-like minor groove widths of 9 -10 Å .…”

“…The crystal structures, on the other hand, tend to have A-like minor groove widths of 9 -10 Å . 13,16,26 This report focuses on the questions: what specific features of the hybrid RNA/DNA PPT make it resistant to RNase H digestion, and how does RNase H recognize the proper 5 0 -cleavage site to define adequately the 5 0 -end of the PPT? In the RT-hybrid complex, the "unzipping" of the 5 0 A-tract 5 and a narrower minor groove in the A-tracts 5,21,27 have been reported as features of the PPT important in recognition.…”

Mutations in the RNase H Primer Grip Domain of Murine Leukemia Virus Reverse Transcriptase Decrease Efficiency and Accuracy of Plus-Strand DNA Transfer

“…Alternatively, the MLV RNase H primer grip mutants could be better at copying through the DNA-RNA junction when it is in a duplex with an RNA strand rather than a DNA strand. The crystal structure of the HIV-1 hybrid duplex formed by a chimeric DNA-tRNA strand and template RNA strand has been reported to have an A-form structure with minor structural perturbations at the r(cpa) ⅐ d(TpG) base pair step (28). Intriguingly, HIV-1 RNase H primer grip mutants have been shown to significantly affect initiation of viral DNA synthesis but not plus-strand DNA transfer (17).…”