Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme
- 29 January 2008
- journal article
- Published by Cold Spring Harbor Laboratory in RNA
- Vol. 14 (3) , 503-513
- https://doi.org/10.1261/rna.622108
Abstract
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 ∼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.Keywords
This publication has 38 references indexed in Scilit:
- A microRNA detection system based on padlock probes and rolling circle amplificationRNA, 2006
- Mechanism of Action of RNase TPublished by Elsevier ,2002
- The Physiological Role of RNase T Can Be Explained by Its Unusual Substrate SpecificityJournal of Biological Chemistry, 2002
- Phage ø29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3′-5′ exonuclease active siteJournal of Molecular Biology, 2000
- A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type φ29 DNA polymerase 1 1Edited by A. R. FershtJournal of Molecular Biology, 1999
- Mutational analysis of ø29 DNA polymerase residues acting as ssDNA ligands for 3′-5′ exonucleolysisJournal of Molecular Biology, 1998
- Structural principles for the inhibition of the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioatesJournal of Molecular Biology, 1998
- SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modelingElectrophoresis, 1997
- CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choiceNucleic Acids Research, 1994
- Metal activation of synthetic and degradative activities of .vphi.29 DNA polymerase, a model enzyme for protein-primed DNA replicationBiochemistry, 1992