Determining Steady-State Kinetics of DNA Polymerase Nucleotide Incorporation
Polymerase enzymes catalyze the replication of DNA by incorporating deoxynucleoside monophosphates (dNMPs) into a primer strand in a 5′ to 3′ direction. Monitoring kinetic aspects of this catalytic process provides mechanistic information regarding polymerase-mediated DNA synthesis and the influences of nucleobase structure. For example, a range of polymerases have different capacities to synthesize DNA depending on the structure of the inserted dNMP (natural or synthetic) and also depending on the templating DNA base (modified vs. unmodified). Under steady-state conditions, relative rates depend on the deoxynucleoside triphosphate (dNTP) residence times in the ternary (polymerase-DNA-dNTP) complex. This chapter describes a method to measure steady-state incorporation efficiencies by which polymerase enzymes insert dNMPs into primer-template (P/T) oligonucleotides. The method described involves the use of a primer oligonucleotide 5′ radiolabeled with [γ-32P]ATP. Significant established applications of this experiment include studies regarding mechanisms of nucleotide misincorporation as a basis of chemically induced DNA mutation. Further, it can provide information important in various contexts ranging from biophysical to medical-based studies.
Key wordsDNA polymerase Steady-state enzyme kinetics Primer extension assay Nucleoside triphosphates
This work was supported by the European Research Council (260341) and the Swiss National Science Foundation (156280). We would like to thank Professors F. Peter Guengerich and Robert Eoff for sharing with us their knowledge and technical expertise of the experiments described here.
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