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# DNA polymerase: Kinetics and DNA replication

## Problem

DNA polymerase operates with fairly high fidelity, meaning that it is able to correctly discriminate between nucleoside triphosphates and ensure that the correct match most often occurs. Two experiments were performed to investigate the kinetics and energetics of this highly specific and coordinated process.
Experiment 1
To evaluate insertion activity, researchers used Drosophila DNA polymerase α, a four-subunit complex that contains polymerase activity but is devoid of 3’ exonuclease activity. Researchers tested the kinetics of Drosophila DNA polymerase α when the enzyme attempted to extend a new DNA strand after a mismatch had already occurred. Researchers used four different deoxyribose primers all labeled with 32P at the 5’ end. Each primer contained an identical nucleotide sequence except for the last nucleotide on the 3’ end. This last nucleotide on the different primers was either adenine (A), guanine (G), thymine (T), or cytosine (C); in Figure 1 “N” represents either A, G, T, or C.
Individually the primers were combined into separate test tubes along with Drosophila DNA polymerase α, an excess of dTTP, pyrophosphatase (the enzyme that cleaves diphosphates), and a short template that contained a nucleotide sequence complementary to the primer’s sequence so there was perfect base pair matching except for the last 3’ nucleotide. This experiment, which allowed the primer to bond to the template DNA molecule, measured the kinetics for the insertion of thymine (T) after a mismatch occurred between the primer and template. Figure 2 shows the velocity vs. concentration curves for this insertion of thymine after a mismatch of G, T, or C.
Experiment 2
A follow up experiment explored the melting temperature of four different short pieces of double stranded DNA (Figure 3), where N is either A, C, G, or T. From the melting temperature results, researchers extrapolated the thermodynamic results produced in Table 1.
Sources: Petruska, J., Goodman, M. F., Boosalis, M. S., Sowers, L. C., Cheong, C., and Tinoco, I., Jr. (1988) Comparison between DNA melting thermodynamics and DNA polymerase fidelity, Proceedings of the National Academy of Sciences of the United States of America 85, 6252-6256.
Based on the graph in Figure 2, for which of the mismatched nucleotide triphosphates does D, r, o, s, o, p, h, i, l, a DNA polymerase α have the highest Km value?