Klumpp K, Doan L, Roberts N A, Handa B
Roche Discovery Welwyn, 40 Broadwater Road, Welwyn Garden City, Herts AL7 3AY, United Kingdom.
J Biol Chem. 2000 Mar 3;275(9):6181-8. doi: 10.1074/jbc.275.9.6181.
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'-hydroxy 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.
流感病毒聚合酶复合物含有一种金属离子依赖性内切核酸酶活性，该活性可从带帽RNA前体产生短的带帽RNA引物分子。先前的研究为RNA切割的双金属离子机制提供了证据，数据与二价金属离子与催化水分子的直接相互作用一致。为了完善这个活性位点的模型，我们生成了一系列DNA、RNA和DNA-RNA嵌合分子，以研究2'-羟基在核酸内切酶核酸底物上的作用。如果核酸底物在5'-端含有帽结构（m7GpppG），我们可以观察到不含任何2'-羟基的核酸底物的特异性切割。带帽的DNA内切核酸酶产物可作为流感病毒聚合酶转录起始的引物。与带帽的RNA底物相比，带帽的DNA底物的表观切割速率大约低5倍。通过将切割位点紧邻5'和3'的脱氧核糖部分替换为核糖部分，DNA底物的切割速率可以提高到RNA水平。同样，通过在切割位点将相同的两个核糖基团与脱氧核糖基团交换，RNA底物的切割速率可以降低到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会影响整体水解速率。观察到的DNA和RNA底物的相对切割速率与催化金属离子与核酸内切酶活性位点中的2'-羟基直接相互作用的观点相悖。
