Duality of polynucleotide substrates for Phi29 DNA polymerase:: 3′ → 5′ RNase activity of the enzyme
Duality of polynucleotide substrates for Phi29 DNA polymerase:: 3′ → 5′ RNase activity of the enzyme
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DOI：
10.1261/rna.622108
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发表时间：
2008-03-01
期刊：
影响因子：
4.5
通讯作者：
Janulaitis, Arvydas
中科院分区：
文献类型：
--
作者：
Lagunavicius, Arunas;Kiveryte, Zivile;Janulaitis, Arvydas
Phi 29 DNA聚合酶是一种依赖于DNA的小分子DNA聚合酶，属于真核生物B型DNA聚合酶。尽管体积小，聚合酶是一种多功能的校对精通酶。它催化两种合成反应（Phi 29末端蛋白的聚合和脱氧核苷酸化），并具有两种降解活性（焦磷酸解和3 ' -> 5 ' DNA外切核酸水解活性）。在这里，我们报告了Phi 29 DNA聚合酶外切降解ssRNA。RNA酶活性以3 '至5 '极性起作用。保守的核酸外切位点（D12 A/D 66 A）中的丙氨酸取代使该酶的RNA酶活性失活，表明单个活性位点负责切割两种底物：DNA和RNA。然而，RNA水解的效率比DNA低10倍。Phi 29 DNA聚合酶广泛用于滚环扩增（RCA）实验。我们证明了该酶的核糖核酸外切酶活性可用于将靶RNA转化为RCA的引物，从而扩大了这种多功能酶的应用潜力，并为RNA检测开辟了新的机会。
Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3 ' -> 5 ' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3 ' to 5 ' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is similar to 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.