Examining Ty3 Polypurine Tract Structure and Function by Nucleoside Analog Interference

Abstract

We have combined nucleoside analog interference with chemical footprinting, thermal denaturation, NMR spectroscopy, and biochemical studies to understand recognition of the polypurine tract (PPT) primer of the Saccharomyces cerevisiae long terminal repeatcontaining retrotransposon Ty3 by its cognate reverse transcriptase. Locked nucleic acid analogs, which constrain sugar ring geometry, were introduced pairwise throughout the PPT (؊)-DNA template, whereas abasic tetrahydrofuran linkages, which lack the nucleobase but preserve the sugar phosphate backbone, were introduced throughout the (؊)-strand DNA template and (؉)-strand RNA primer. Collectively, our data suggest that both the 5-and 3-portions of the PPT-containing RNA/DNA hybrid are sensitive to nucleoside analog substitution, whereas the intervening region can be modified without altering cleavage specificity. These two regions most likely correspond to portions of the PPT that make close contact with the Ty3 reverse transcriptase thumb subdomain and RNase H catalytic center, respectively. Achieving a similar phenotype with nucleoside analogs that have different effects on duplex geometry reveals structural features that are important mediators of Ty3 PPT recognition. Finally, the results from introducing tetrahydrofuran lesions around the scissile PPT/unique 3-sequence junction indicate that template nucleobase ؊1 is dispensable for catalysis, whereas a primer nucleobase on either side of the junction is necessary.Although reverse transcription in long terminal repeat (LTR) 3 -containing retrotransposons of Saccharomyces cerevisiae (Ty1 and Ty3) and Saccharomyces paradoxus (Ty5) can be likened to that in retroviruses, notable differences have recently been documented. These include (a) initiation of (Ϫ)-strand DNA synthesis from an internal site of the host-coded tRNA primer as opposed to its 3Ј terminus (1), (b) initiation of (Ϫ)-strand DNA synthesis via self-priming (2), (c) use of a bipartite primer-binding site derived from each end of the viral RNA genome to initiate (Ϫ)-strand DNA synthesis (3), and (d) divergence in size and sequence of the (ϩ)-strand polypurine tract (PPT) primers (see Fig. 1) (4, 5). A detailed biochemical study of these processes has been hampered by the lack of purified LTR-containing retrotransposon reverse transcriptases (RTs). However, the availability of active recombinant Ty1 and Ty3 enzymes (6, 7) now allows interactions with their cis-acting signals to be compared and contrasted with those of the retroviral counterparts.A focus of our recent studies (8 -13) has been understanding the mechanism that positions RT on the PPT-containing RNA/DNA hybrid for accurate cleavage at the PPT/unique 3Ј-sequence (U3) junction. Such specificity is required to create the (ϩ)-strand primer and later to remove it from nascent (ϩ)-DNA, thereby creating an integration-competent, double-stranded DNA provirus. Surprisingly, although a compilation of PPT sequences (14, 15) suggests a common "modular" make-up of homopolymeric rA:dT and rG:dC blo...

Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis

“…To explore this notion, we have inserted a variety of nucleoside analogs into PPT-containing RNA/DNA hybrids to alter their structure in a manner that minimally alters sequence context. Examples include pyrimidine isosteres, or shape mimics, which locally remove hydrogen bonding (12,17), abasic tetrahydrofuran linkages, which remove the nucleobase while preserving the sugar-phosphate backbone (18,19), pyrrolo-dC, a fluorescent cytosine analog providing information on local base pairing (20) and locked nucleic acid monomers, which locally stabilize the phosphate backbone. This strategy demonstrated (i) that the junction between the rG:dC and upstream rA:dT tract is particularly sensitive to altering hydrogen bonding (17) (ii) weakened base pairing at position −2 (20) and (iii) that DNA nucleobases on either side of the PPT-U3 junction are not required for accurate cleavage (18).…”

Examining Interactions of HIV-1 Reverse Transcriptase with Single-stranded Template Nucleotides by Nucleoside Analog Interference

“…Based on this model, site-directed mutagenesis has demonstrated that substituting Phe-61 alters fidelity (34), processivity, and strand displacement synthesis (35). The focus of the present study was to determine whether (a) altering the nucleobase, sugar, or phosphate backbone of the 5Ј template overhang ahead of the polymerase catalytic center influences orientation of the template base and (b) how this is affected by substitution of amino acids implicated in such interactions.The strategy we pursued is based on our recent analysis of (ϩ) strand DNA synthesis from the polypurine tract primer of retroviruses (25,26,28) and long terminal repeat-containing retrotransposons (36,37). In these studies nucleoside analogs altering duplex geometry were exploited to define regions of the polypurine tract-containing RNA/DNA hybrid interacting with the cognate RT.…”

“…The strategy we pursued is based on our recent analysis of (ϩ) strand DNA synthesis from the polypurine tract primer of retroviruses (25,26,28) and long terminal repeat-containing retrotransposons (36,37). In these studies nucleoside analogs altering duplex geometry were exploited to define regions of the polypurine tract-containing RNA/DNA hybrid interacting with the cognate RT.…”

Probing Anomalous Structural Features in Polypurine Tract-Containing RNA−DNA Hybrids with Neomycin B

“…If the sites were functionally independent, each should exhibit a binding affinity similar to that of PPT wt . While coupling between NB binding sites on the Ty3 PPT was unexpected, propagation of structural signals has been observed along the rA·rG·rA symmetric steps of the PPT with both dF base analogue (9) and LNA-substituted PPTs (36). …”