Duality of polynucleotide substrates for Phi29 DNA polymerase:: 3′ → 5′ RNase activity of the enzyme

被引：19

作者：

Lagunavicius, Arunas ^{[1
]}

Kiveryte, Zivile ^{[1
]}

Zimbaite-Ruskuliene, Vilma ^{[1
]}

Radzvilavicius, Tomas ^{[1
]}

Janulaitis, Arvydas ^{[1
]}

机构：

[1] Fermentas UAB, LT-02241 Vilnius, Lithuania

来源：

RNA | 2008年 / 14卷 / 03期

关键词：

Phi29 DNA polymerase; exoribonuclease; RNase; rolling circle amplification; RCA;

D O I：

10.1261/rna.622108

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：503 / 513

页数：11

共 38 条

[1] Signal amplification of padlock probes by rolling circle replication [J].