Nonpolar thymine isosteres in the Ty3 polypurine tract DNA template modulate processing and provide a model for its recognition by Ty3 reverse transcriptase
Nonpolar thymine isosteres in the Ty3 polypurine tract DNA template modulate processing and provide a model for its recognition by Ty3 reverse transcriptase
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DOI：
10.1074/jbc.m302374200
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发表时间：
2003-07-18
影响因子：
4.8
通讯作者：
Le Grice, SFJ
中科院分区：
文献类型：
--
作者：
Lener, D;Kvaratskhelia, M;Le Grice, SFJ
尽管序列和大小不同，逆转录病毒和含有长末端重复序列的逆转录转座子的多嘌呤段（PPT）引物通过其同源逆转录酶（RT）从（+）U3 RNA和DNA准确加工。在本文中，我们证明了人类免疫缺陷病毒-1 PPT上Ty 3逆转录转座子RT的错位导致不精确地去除相邻的（+）-RNA和未能从引物释放（+）-DNA。基于这些观察，我们通过化学合成RNA/DNA杂合体来探索Ty 3 PPT识别的结构基础，所述RNA/DNA杂合体的（+）- DNA模板被非氢键胸腺嘧啶等排体2，4-二氟-5-甲苯（F）取代。我们观察到T3 F取代位点与增强的核糖核酸酶H（RNaseH）活性之间存在一致的空间相关性，类似于下游12 -13 bp。在最明显的情况下，PPT位置-1/-2处的双重T3 F取代将RNase H切割几乎完全重定向到新位点。这个不寻常的碱基的结构特征表明，它插入到Ty 3 PPT（-）- DNA模板削弱双链体，诱导不稳定，这是由Ty 3 RT的结构元件识别，类似于12 -13 bp从其RNase H催化中心。这种相互作用的可能候选者是拇指亚结构域，其小沟结合道最有可能接触双链体。由T → F取代衍生的空间关系还推断Ty 3 PPT加工需要识别其紧邻5'的序列，从而将RNase H催化中心定位在PPT-U3连接上，这一概念通过本文的额外诱变研究得到加强。
Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (+) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (+)-RNA and failure to release (+)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (+)- DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene ( F). We observed a consistent spatial correlation between the site of T 3 F substitution and enhanced ribonuclease H ( RNase H) activity similar to12-13 bp downstream. In the most pronounced case, dual T 3 F substitution at PPT positions -1/-2 redirects RNase H cleavage almost exclusively to the novel site. The structural features of this unusual base suggest that its insertion into the Ty3 PPT (-)- DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT similar to12-13 bp from its RNase H catalytic center. A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex. The spatial relationship derived from T-->F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5' vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper.