Proofreading activity of Pfu thermostable DNA polymerase on a 6-O-Methylguanine Containing Template Monitored by ESI-FTICR Mass Spectrometry.
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.
- Research Organization:
- Pacific Northwest National Lab., Richland, WA (US), Environmental Molecular Sciences Laboratory (US)
- Sponsoring Organization:
- US Department of Energy (US)
- DOE Contract Number:
- AC06-76RL01830
- OSTI ID:
- 15008817
- Report Number(s):
- PNNL-SA-40946; 3171; TRN: US200427%%244
- Journal Information:
- Chembiochem, Vol. 5, Issue 7; Other Information: PBD: 5 Jul 2004
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
AMPLIFICATION
DNA
DNA DAMAGES
DNA POLYMERASES
ENZYMES
GUANINE
ION CYCLOTRON-RESONANCE
IONIZATION
MASS SPECTROSCOPY
POLYMERASE CHAIN REACTION
POLYMERASES
POLYMERIZATION
ENVIRONMENTAL MOLECULAR SCIENCES LABORATORY
MASS SPECTROMETRY
DNA BASE MODIFICATION
PCR
EXONUCLEASE ACTIVITY