Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.

Xi-Peng Liu,Jian-Hua Liu

doi:10.1371/journal.pone.0027248

Abstract

Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55–65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.

Highlights

Deamination of cytosine in DNA leads to dU/dG damage and deamination of dCTP results in dUTP that can be misincorporated into the genome during replication in the form of dUMP
The optimal reaction of dU removal by A. pernix UDG (ApeUDG) The glycosylase activity of ApeUDG was characterized in the reaction buffers with pH values ranging from 5.5 to 10.5
Our results shows that the use of thermostable ApeUDG largely improves the yield of PCR by Pfu DNA polymerase, suggesting that the activity of Pfu DNA polymerase is blocked by dU in DNA generated during the PCR reaction, and that ApeUDG can efficiently remove dU from DNA (Fig. 5A)

Summary

Introduction

Deamination of cytosine in DNA leads to dU/dG damage and deamination of dCTP results in dUTP that can be misincorporated into the genome during replication in the form of dUMP. The high temperature rate constants for the spontaneous deamination of cytosine in DNA and dCTP are several orders of magnitude higher than those at more moderate temperatures [1]. The family B DNA polymerase binds to the mutant-base dU in the template-strand and stops ahead of dU damage, avoiding the incorporation of dAMP opposite to dU [7,8,9]. These proteins are thought to be the main participants in the processing of dU damage

Methods

Results

Conclusion