DNA replication: Kinetics and thermodynamics of base mismatch

DNA polymerase operates with high fidelity, accurately discriminating between nucleoside triphosphates to ensure correct base pairing. Two experiments were conducted to investigate the kinetics and energetics resulting from experimentally introduced mismatched base pairs.

Researchers tested the kinetics of Drosophila DNA polymerase α, a four-subunit complex devoid of 3’ exonuclease activity, when it attempted to extend a new DNA strand. To introduce the mismatch, researchers used four different deoxyribose primers all labeled with 32P at their 5’ ends. Each primer contained an identical nucleotide sequence except for the last nucleotide at the 3’ end. Figure 1 shows the primer and template used to measure the enzyme's rate of extension; the rate of extension (i.e. the insertion of T opposite A in the template) was compared between matched and mismatched 3' primer termini.

Figure 1 DNA primer and template used; N = A, C, T, or G

The primers were combined into separate test tubes along with Drosophila DNA polymerase α, dTTP, pyrophosphatase (an enzyme that cleaves diphosphates), and the DNA template. The velocity–concentration curves for thymine insertion after each mismatch are depicted in Figure 2.

Figure 2 Velocity vs. dTTP concentration for each mismatched primer–template terminus

Researchers studied the melting temperatures of the last nine base pairs of the primer–template duplexes (shown in Figure 3). From these results, researchers extracted the thermodynamic data that are provided in Table 1.

Figure 3 Short DNA sequences studied; N = A, C, G, or T

Table 3 Thermodynamics of DNA melting