Kinetics and thermodynamics of base mismatch
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Kinetics and thermodynamics of base mismatch
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[post_content] => Practice Passage (Question 1-6)
*This passage is the property of Khan Academy and has been reformatted into an AAMC-style interface in their entirety by MedLife Mastery. MedLife Mastery does not endorse and is not an affiliate of Khan Academy.
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.
Experiment 1
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
Experiment 2
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
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.
[post_title] => Kinetics and thermodynamics of base mismatch
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[question] => Which of the following is NOT consistent with the experimental setup and results of Experiment 1?
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[answer] => 2
[description] => Reason for Correct Answer:
The data in Figure 2 show the effects of the base mismatches on the ability of DNA polymerase α to extend the new DNA strand.
The data show that DNA polymerase performed the best under the G–T mismatch conditions; you can see that the overall reaction velocities and the Vmax were higher in this condition.
The data also show that the thymidine–thymidine mismatch created the lowest affinity (highest Km) of the enzyme for the substrate. The Km value (which equals the substrate concentration at ½ Vmax) appears to be the greatest.
In the experimental setup, the reaction mixtures only contain dTTP. This means that the primer (with the template as shown) will only be extended by one base pair.
Choice B is the only statement that is incorrect. The dTTP added to the reaction mixture forms the base pair (with A) AFTER the N–T mismatch is already introduced by the primer.
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[each_answer] => A. The primer is only extended by one nucleotide in the experimental setup described.
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[each_answer] => B. The inclusion of dTTP in the reaction mixtures helps increase the frequency of N–T mismatches.
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[2] => Array
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[each_answer] => C. A thymidine–thymidine mismatch most dramatically increases the Kₘ of DNA polymerase α for its substrate.
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[each_answer] => D. The guanine–thymidine mismatch has the least impact on the performance of DNA polymerase α.
)
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[1] => Array
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[quiz_unique_key] => 3243476205
[question] => What is the most likely reason researchers used Drosophila DNA polymerase α?
[value] => Array
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[answer] => 2
[description] => Reason for Correct Answer:
Withstanding large heat changes is important for PCR, but here the researchers were just investigating extension kinetics. There is no need to ensure the polymerase can withstand large heat changes.
There is no information in the passage to suggest these experiments were designed to explain phenomena in human DNA polymerase.
DNA polymerase exonuclease function proofreads and repairs newly synthesized DNA strands by removing mismatched nucleotides, ensuring accurate DNA replication.
Therefore, the lack of exonuclease function in DNA polymerase α ensured that it could not fix the mismatched bases that the researchers were trying to study.
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[answers] => Array
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[0] => Array
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[each_answer] => A. Drosophila DNA polymerase α is able to withstand large heat changes.
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[each_answer] => B. Drosophila DNA polymerase α is unable to proofread the new DNA.
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[2] => Array
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[each_answer] => C. Drosophila DNA polymerase α is very similar to human DNA polymerase.
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[each_answer] => D. All the genes for Drosophila DNA polymerase α are known and can be manipulated.
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[2] => Array
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[quiz_unique_key] => 2187790141
[question] => The data in Table 1 best supports which conclusion?
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[answer] => 2
[description] => Reason for Correct Answer:
Table 1 shows the thermodynamic data for separating, or melting, the DNA strands/base pairings (note that in the context of DNA, melting does not refer to the change of state from solid to liquid, but to the separation of the two component strands). All values would have opposite signs for the bonding, or annealing, of the strands.
In terms of favorability, an overall favorable reaction is characterized by a negative change in Gibbs free energy (−ΔG). A negative enthalpy change (−ΔH) is favorable and a positive entropy (+ΔS) change is favorable. [Remember, ΔG = ΔH − TΔS.]
The table shows that melting, or separating, the DNA strands is unfavorable overall (+ΔG), and it is unfavorable in terms of enthalpy change (+ΔH) but favorable in terms of entropy change (+ΔS). So, it’s not Choice C.
This means that the opposite process – the annealing of DNA strands and their bases – has the opposite characteristics, including an unfavorable entropy change (−ΔS). So, it’s not Choice D.
The magnitude of ΔG is a measure of how favorable a process is at a given temperature; processes with higher ΔG values are less favorable than those with lower ΔG values.
Because the process in question is the separation of DNA strands that differ by only one base pair, a less favorable process (larger ΔG) indicates a terminal base pair with higher thermodynamic stability (the base pair is harder to separate).
Because the ΔG values for separation with A–T and G–T base pairs (the purines) are higher than the corresponding value for the T–T base pair, base pairs between purines and thymine are thermodynamically more stable than a T–T base pair.
Note that no conclusions can be made about the thermodynamic stability of base pairs between adenosine (A) and other bases; in this experiment, only T is paired with different bases.
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[each_answer] => A. An A–T base pair is thermodynamically more stable than an A–C base pair.
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[each_answer] => B. Base pairs between purines and thymine are thermodynamically more stable than a T–T base pair.
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[2] => Array
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[each_answer] => C. In terms of enthalpy, it is more favorable to separate DNA strands than to bond them.
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[each_answer] => D. The formation of a T–T base pair is entropically favorable.
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[3] => Array
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[quiz_unique_key] => 1579828684
[question] => Why did the researchers most likely add pyrophosphatase to the reaction vessels in Experiment 1?
[value] => Array
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[answer] => 3
[description] => Reason for Correct Answer:
The passage states that pyrophosphatase is an enzyme that cleaves diphosphates (i.e. it cleaves “pyrophosphate,” which is two bonded phosphates).
During DNA replication, polymerase adds dinucleotide triphosphates to the growing DNA strand. During the incorporation of a nucleotide into the growing DNA strand, a pyrophosphate group is released.
By adding pyrophosphatase to the reaction mixture, the pyrophosphate groups generated during the addition of dTTP can be rapidly converted into inorganic phosphate ions.
The fact that this conversion is essential suggests that the accumulation of pyrophosphate can inhibit the DNA synthesis reaction. Removing pyrophosphate ensures that the DNA polymerase reaction can proceed smoothly, allowing the researchers to specifically focus on the extension of matched and mismatched 3′ primer termini without interference from inhibitory pyrophosphate molecules.
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[answers] => Array
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[0] => Array
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[each_answer] => A. Pyrophosphatase prepares dinucleoside triphosphates (dNTPs) for incorporation into DNA by cleaving pyrophosphate.
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[each_answer] => B. Pyrophosphatase helps dinucleoside triphosphates (dNTPs) to bind to the growing DNA strand.
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[each_answer] => C. The accumulation of pyrophosphate can inhibit the DNA synthesis reaction.
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[each_answer] => D. The researchers wanted to measure the kinetics of pyrophosphate breakdown.
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[quiz_unique_key] => 1498560436
[question] => In Experiment 2, what type of bonds are formed or broken?
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[answer] => 3
[description] => Reason for Correct Answer:
In Experiment 2 the strands of DNA are separated when the temperature is raised. The temperature at which the strands separate is called the melting temperature.
The bonds keeping the strands of DNA together are between the base pairs.
Hydrogen bonds form between DNA base pairs; these are broken when the strands of DNA separate.
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[answers] => Array
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[0] => Array
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[each_answer] => A. Covalent bonds
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[each_answer] => B. Ionic bonds
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[each_answer] => C. Hydrogen bonds
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[each_answer] => D. Metallic bonds
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[5] => Array
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[quiz_unique_key] => 3044259542
[question] => Fully complementary DNA duplexes were formed from each of the DNA strands below (no mismatched base pairs were present in any of the duplexes). Which duplex would be expected to have the highest melting temperature?
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[answer] => 4
[description] => Reason for Correct Answer:
As discussed in the previous explanation, DNA strands are held together by hydrogen bonds between complementary nucleotide bases. Specifically, adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G). These base pairs are connected with hydrogen bonds.
The more hydrogen bonds that must be broken, the higher the melting temperature of the DNA duplex.
For this reason, longer DNA duplexes, like those shown in Choices C and D, tend to have higher melting temperatures than those shown in Choices A and B.
Next, you need to look at the composition of the bases. Notice that adenine forms two hydrogen bonds with thymine, and cytosine forms three hydrogen bonds with guanine.
https://commons.wikimedia.org/wiki/File:Figure_02_03_08.jpg
Therefore, molecules with a higher proportion of C–G pairs, compared with A–T pairs, will have a higher melting temperature. Accordingly, Choice D is the answer. Notice that Choice D shows only one strand of the DNA molecule in question, but the final DNA molecule will primarily have C–G pairs; the DNA strand in Choice D has 9 nucleotides out of 15 that are C or G, whereas the strand in Choice C has only 6.
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[each_answer] => A. TATCGGTTC
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[each_answer] => B. CGCCTAGGC
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[each_answer] => C. TATCGGTTCAAGTCT
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[each_answer] => D. CGCCTAGGCAACTGT
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[553808|1] => B
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[553808|6] => D
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Figure 1
Figure 2
Figure 3 
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.
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