RNA and DNA hydrolysis are catalyzed by the influenza virus endonuclease
RNA and DNA hydrolysis are catalyzed by the influenza virus endonuclease
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DOI：
10.1074/jbc.275.9.6181
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发表时间：
2000-03-03
影响因子：
4.8
通讯作者：
Handa, B
中科院分区：
文献类型：
--
作者：
Klumpp, K;Doan, L;Handa, B
流感病毒聚合酶复合物含有金属离子依赖性内切核酸酶活性，其从加帽RNA前体产生短加帽RNA引物分子。先前的研究已经为RNA切割的双金属离子机制提供了证据，并且数据与二价金属离子与催化水分子的直接相互作用一致。为了完善该活性位点的模型，我们已经产生了一系列DNA、RNA和DNA/RNA嵌合分子来研究核酸内切酶的核酸底物上的2 '-羟基基团的作用。如果核酸底物在5'端含有帽结构（m7 GpppG），我们可以观察到没有任何2 '-羟基基团的核酸底物的特异性切割。加帽的DNA内切酶产物作为流感病毒聚合酶转录起始的引物起作用。与加帽的RNA底物相比，加帽的DNA底物的表观切割速率低约5倍。通过用核糖基部分取代紧邻切割位点5'和3'的脱氧核糖基部分，可以将DNA底物的切割速率提高到RNA水平。类似地，通过在切割位点用脱氧核糖基交换相同的两个核糖基，可以将RNA底物的切割速率降低到DNA水平。这些结果表明，2 '-羟基对于流感病毒核酸内切酶结合和切割核酸不是必需的，但核酸内切酶活性位点中核酸构象的微小差异可影响总水解速率，所观察到的与DNA和RNA底物的相对切割速率反对催化金属离子与2 '-在核酸内切酶活性位点的羟基基团。
The influenza virus polymerase complex contains a metal ion-dependent endonuclease activity, which generates short capped RNA primer molecules from capped RNA precursors. Previous studies have provided evidence for a two-metal ion mechanism of RNA cleavage, and the data are consistent with a direct interaction of a divalent metal ion with the catalytic water molecule. To refine the model of this active site, we have generated a series of DNA, RNA, and DNA RNA chimeric molecules to study the role of the 2'-hydroxy groups on nucleic acid substrates of the endonuclease, We could observe specific cleavage of nucleic acid substrates devoid of any 2'-hydroxy groups if they contained a cap structure (m7GpppG) at the 5'-end. The capped DNA endonuclease products were functional as primers for transcription initiation by the influenza virus polymerase, The apparent cleavage rates were about 5 times lower with capped DNA substrates as compared with capped RNA substrates. Cleavage rates with DNA substrates could be increased to RNA levels by substituting the deoxyribosyl moieties immediately 5' and 3' of the cleavage site with ribosyl moieties. Similarly, cleavage rates of RNA substrates could be lowered to DNA levels by exchanging the same two ribosyl groups with deoxyribosyl groups at the cleavage site. These results demonstrate that the 2'-hydroy groups are not essential for binding and cleavage of nucleic acids by the influenza virus endonuclease, but small differences of the nucleic acid conformation in the endonuclease active site can influence the overall rate of hydrolysis, The observed relative cleavage rates with DNA and RNA substrates argue against a direct interaction of a catalytic metal ion with a 2'-hydroxy group in the endonuclease active site.