Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3′ polypurine tract
Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3′ polypurine tract
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DOI：
10.1093/nar/gkh307
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发表时间：
2004-02-01
影响因子：
14.9
通讯作者：
Le Grice, SFJ
中科院分区：
文献类型：
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作者：
Dash, C;Rausch, JW;Le Grice, SFJ
最近的结构分析表明，含有 HIV-1 多嘌呤束 (PPT) 的 RNA/DNA 杂交体中存在异常碱基配对的局部区域，并且这些扭曲可能在 PPT 功能中发挥作用。为了直接检查这一点，我们将吡咯并脱氧胞嘧啶 (pdC)（一种脱氧胞嘧啶 (dC) 的荧光环境敏感类似物）引入到含有 PPT 的杂交体的 DNA 链中。这些杂交体的稳态荧光分析表明，即使在没有逆转录酶 (RT) 的情况下，PPT-U3 连接处的 DNA 碱基 11 nt 也是不配对的。在双链体下游部分的 (rG:dC)(6) 区域内也观察到不稳定的碱基配对，这表明 HIV-1 RT 可能在 PPT 选择过程中识别多个预先存在的扭曲。 HIV-1 RT 主要在 PPT-U3 连接处水解含有 pdC 的杂合体，表明该类似物不会引起双链体的总体结构变形。然而，在距 pdC 取代位点 3 bp 处经常观察到异常切割，很可能反映了 RNase H 引物夹内类似物和氨基酸残基之间的特定相互作用。 DNA 双链体模板链内的 pdC 取代似乎不会显着影响 RT 催化的 DNA 合成。讨论了这些发现对使用 pdC 检查核酸结构的影响。
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.