Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: Discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p
Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: Discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p
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DOI：
10.1016/j.jmb.2004.02.059
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发表时间：
2004-04-23
影响因子：
5.6
通讯作者：
Abou Elela, S
中科院分区：
文献类型：
--
作者：
Lamontagne, B;Hannoush, RN;Abou Elela, S
已知双链RNA（dsRNA）特异性RNase III家族的成员使用保守的dsRNA结合结构域（dsRBD）来区分RNA A型螺旋和DNA B型螺旋，然而，这种选择性的基础及其对切割特异性的影响仍然未知。在这里，我们直接研究了芽殖酵母RNase III（Rnt 1 p）对dsRNA识别和切割的分子要求，并将其与细菌RNase III和裂殖酵母RNase III（Pac 1）进行了比较。我们合成的底物与化学修饰的核苷酸附近，切割位点，或与不同的DNA/RNA组合，并研究其结合和切割Rnt 1 p。用2 '-脱氧-2'-氟-β-D-核糖（2' F-RNA）、脱氧核糖核苷酸或2'-O-甲基核糖核苷酸取代易断裂的磷酸二酯键附近的核糖核苷酸，允许被Rnt 1 p切割，而引入2，V-磷酸二酯键允许结合，但不允许切割。这表明磷酸二酯键相对于核酸酶结构域的位置，而不是2 '-OH基团，对Rnt 1 p的切割至关重要。令人惊讶的是，Rnt 1 p结合的DNA螺旋与NGNN四核糖核苷酸环，表明至少一个成员的RNase III家族的结合不限于RNA。结果还表明，dsRBD可以容纳B型DNA双链体。有趣的是，Rnt 1 p，而不是Pac 1或细菌RNase III，切割DNA/RNA杂交体的DNA链，表明A型RNA螺旋不是Rnt 1 p切割所必需的。与此相反，RNA/DNA杂交体结合到Rnt 1 p上，但不被Rnt 1 p切割，这强调了位于四环3'端的核苷酸的关键作用，并表明底物识别的不对称模式。在细胞提取物中，天然酶有效地切割了DNA/RNA杂交体，表明Rnt 1 p底物特异性比以前认为的要广泛得多。这种新的RNA依赖性脱氧核糖核酸酶活性的发现在设计新的靶向活跃转录的DNA的抗病毒策略方面具有潜在的意义。(C)2004爱思唯尔有限公司保留所有权利。
Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near, the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-D-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2, V-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA. (C) 2004 Elsevier Ltd. All rights reserved.