July 5, 2004
Journal Article

Proofreading activity of Pfu thermostable DNA polymerase on a 6-O-Methylguanine Containing Template Monitored by ESI-FTICR Mass Spectrometry.

Abstract

Abstract DNA damage can take the form of chemical lesions that interfere with DNA polymerization and therefore, the replication of DNA within a cell. In this report we examine the effect of a particular type of base modification, a 6-O-methyl group on a guanine base. Previous reports using different DNA polymerases have identified an induced base substitution. However, this process has not been studied using polymerase chain reaction (PCR) enzyme. Electrospray ionization (ESI) mass spectrometry (MS) was used to examine the effect of this type of base on the PCR. Using Fourier transform ion cyclotron resonance (FTICR) MS, two types of amplification products were clearly resolved, one corresponding to the expected product composition, and one with a dG-dC to dA-dT base substitution. Further investigation found that the same substitution occurred when amplified with an exonuclease (exo-) form of the polymerase (lacking a proofreading function). This technique provides complementary information to other methods and is a sensitive method detecting effects of DNA damage on enzyme polymerization.

Revised: August 13, 2004 | Published: July 5, 2004

Citation

Wunschel D.S., C.D. Masselon, B. Feng, and R.D. Smith. 2004. Proofreading activity of Pfu thermostable DNA polymerase on a 6-O-Methylguanine Containing Template Monitored by ESI-FTICR Mass Spectrometry. Chembiochem 5, no. 7:1012-1015. PNNL-SA-40946.