Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3' polypurine tract

Abstract

Recent structural analyses indicate that localized regions of abnormal base pairing exist within RNA/DNA hybrids containing the HIV-1 polypurine tract (PPT) and that these distortions may play a role in PPT function. To examine this directly, we have introduced pyrrolo-deoxycytosine (pdC), a fluorescent, environmentally sensitive analog of deoxycytosine (dC), into the DNA strand of PPT-containing hybrids. Steady-state fluorescence analysis of these hybrids reveals that the DNA base 11 nt from the PPT-U3 junction is unpaired even in the absence of reverse transcriptase (RT). Unstable base pairing is also observed within the (rG:dC)6 tract in the downstream portion of the duplex, suggesting that HIV-1 RT may recognize multiple pre-existing distortions during PPT selection. HIV-1 RT hydrolyzes pdC-containing hybrids primarily at the PPT-U3 junction, indicating that the analog does not induce a gross structural deformation of the duplex. However, aberrant cleavage is frequently observed 3 bp from the site of pdC substitution, most likely reflecting a specific interaction between the analog and amino acid residues within the RNase H primer grip. pdC substitution within the template strand of a DNA duplex does not appear to significantly affect RT-catalyzed DNA synthesis. Implications of these findings on the use of pdC to examine nucleic acid structure are discussed.

Figure 1

(A) Structural features of the HIV-1 PPT derived from crystallographic and KMnO₄ footprinting data (10,13). Weak base pairs are represented by solid bars and unpaired bases are enclosed within circles. KMnO₄-sensitive thymines in the DNA template are shaded. The sequences shown reflect those of synthetic oligonucleotides used for biochemical and fluorescence analysis. (B) Structure and hydrogen bonding pattern of dC:dG (left) and pdC:dG base pairs (right).

Figure 2

Fluorescence analysis of pdC-substituted constructs. Emission spectra of DNAs containing pdC at positions (A) –6, (B) +1 and (C) –11 are shown. Measurements of 1 µM single-stranded DNAs and RNA/DNA hybrids are indicated by open squares and open circles, respectively. (D) Quantitative fluorescence quenching map of the PPT-containing hybrid duplex. Positions of pdC substitution are indicated. Relative quenching (%) = 100 × (single-stranded fluorescence – hybrid fluorescence) ÷ single-stranded fluorescence at 460 nM. Bar heights and error bars indicate the average relative quenching (%) and standard error, respectively, derived from three independent experiments.

Figure 3

RNase H cleavage analysis of pdC-substituted HIV-1 PPT constructs. Cleavage at the PPT–U3 junction is designated –1 (see Fig. 1A). Hydrolysis profiles of HIV-1 RT (A) and Ty3 RT (B) are shown. Intact substrate is shown in lane C. Lanes U indicate cleavage of the unsubstituted, PPT-containing RNA/DNA hybrid. Numbering above each lane reflects the sites of pdC substitution within the DNA strand. Asterisks demark products of analog-directed hydrolysis. Note that no such products are indicated in lane –4, since in this case analog-directed cleavage would be expected to occur at the PPT–U3 junction.

Figure 4

Processing of pdC-substituted HIV-1 PPT constructs by primer grip mutants of HIV-1 RT. Cleavage profiles of (A) wild-type p66/p51 (unsubstituted hybrid only) (B) p66^T473A/p51, (C) p66^N474A/p51, (D) p66^Q475A/p51, (E) p66^K476A/p51 and (F) p66^Y501A/p51 HIV-1 RTs are shown, with lane designations as described in Figure 3.

Figure 5

DNA polymerase activities of HIV-1 RT and Ty3 RT on pdC-substituted DNAs. (A) A 5′-³²P-end-labeled DNA primer is annealed to either an unsubstituted DNA template or a template containing a pdC substitution at one of the six positions shaded gray. For clarity, the numbering convention was changed to reflect nucleoside positioning relative to the primer 3′ terminus (e.g. the initial dA incorporation occurs opposite the template dT at position +1). The arrow denotes the direction of DNA synthesis. DNA synthesis catalyzed by (B) HIV-1 RT or (C) Ty3 RT was examined. Unextended primer (P), full-length product (P+22) and products of intermediate length are indicated. Lane designations reflect sites of pdC substitution, with DNA synthesis over an unsubstituted template shown in lane U.

Figure 6

Effects of pdC on fidelity of DNA synthesis catalyzed by HIV-1 RT. (A) The substrates and numbering convention are identical to those described in Figure 5, except that pdC is introduced exclusively at position +2. (B) DNA synthesis profiles from reactions containing (lanes a) dATP and dGTP, (lanes b) dATP and dTTP, (lanes c) dATP and dCTP, (lanes d) dATP alone or (lanes e) all four nucleotides. Unextended primer (P), fully extended primer (P+21), intermediate products and the template used in the reaction (top) are indicated.

Figure 7

HIV-1 RT–PPT/DNA during plus-strand primer selection. Based on the structure reported by Sarafianos et al. (10), the schematic reflects the projected positioning of RT for cleavage at the PPT–U3 junction (open circle), as well as the nucleic acid distortion centered around positions –12/–13. Anticipated locations of DNA polymerase and RNase H active sites, the RNase H primer grip and all residues projected to contact nucleic acid are indicated. PPT nucleotides are indicated in bold.