Passage
DNA polymerization is one of the most conserved mechanisms of genome replication. Synthesis of a complete DNA strand requires a template, primers, a polymerase enzyme, and sufficient deoxyribonucleotide triphosphates (dNTPs) . The DNA polymerase enzyme binds consecutive base pairs on the template strand and extends the double helix by adding dNTPs to the primer. The amino acid residues in the active site of DNA polymerase form hydrogen bonds with Watson-Crick donors and acceptors on incoming DNA nucleotides to facilitate base pairing.The formation of the DNA double helix creates opposing changes in entropy and enthalpy. Favorable bonding interactions via hydrogen bonds during Watson-Crick base pairing results in negative enthalpy, and restricted rotation and flexibility of the DNA backbone generates negative entropy. Scientists hypothesize that hydrogen bonding between bases not only stabilizes the double helix but is also crucial for selective and efficient replication.Analogs that are similar in size and shape to naturally occurring bases can be used to determine the influence of hydrogen bonding on base pair selectivity. To mimic the structure of deoxythymidine triphosphate (dTTP) , researchers synthesized dNTP derivatives of difluorotoluene (dFTP) , a nonpolar analog that lacks Watson-Crick hydrogen bonding. Klenow fragment (KF) polymerase, which has 3′-5′ but not 5′-3′ exonuclease activity, was incubated with a mixture of DNA template, primers, and dNTPs, including dFTP derivatives. The efficiency of dFTP and natural dTTP nucleotide incorporation into a growing primer strand by KF is shown in Figure 1.
Figure 1 Template-specific selection of dFTP and dTTP by the KF enzyme
Adapted from Moran S, Ren RX, Kool ET. A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity. Proc Natl Acad Sci USA. 1997;94(20) :10506-11.
-Based on the information in the passage, what effect would incorporation of dFTP in place of dTTP most likely have on the melting temperature of a DNA molecule of a fixed length?

Question

Passage
DNA polymerization is one of the most conserved mechanisms of genome replication. Synthesis of a complete DNA strand requires a template, primers, a polymerase enzyme, and sufficient deoxyribonucleotide triphosphates (dNTPs) . The DNA polymerase enzyme binds consecutive base pairs on the template strand and extends the double helix by adding dNTPs to the primer. The amino acid residues in the active site of DNA polymerase form hydrogen bonds with Watson-Crick donors and acceptors on incoming DNA nucleotides to facilitate base pairing.The formation of the DNA double helix creates opposing changes in entropy and enthalpy. Favorable bonding interactions via hydrogen bonds during Watson-Crick base pairing results in negative enthalpy, and restricted rotation and flexibility of the DNA backbone generates negative entropy. Scientists hypothesize that hydrogen bonding between bases not only stabilizes the double helix but is also crucial for selective and efficient replication.Analogs that are similar in size and shape to naturally occurring bases can be used to determine the influence of hydrogen bonding on base pair selectivity. To mimic the structure of deoxythymidine triphosphate (dTTP) , researchers synthesized dNTP derivatives of difluorotoluene (dFTP) , a nonpolar analog that lacks Watson-Crick hydrogen bonding. Klenow fragment (KF) polymerase, which has 3′-5′ but not 5′-3′ exonuclease activity, was incubated with a mixture of DNA template, primers, and dNTPs, including dFTP derivatives. The efficiency of dFTP and natural dTTP nucleotide incorporation into a growing primer strand by KF is shown in Figure 1.

Figure 1 Template-specific selection of dFTP and dTTP by the KF enzyme
Adapted from Moran S, Ren RX, Kool ET. A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity. Proc Natl Acad Sci USA. 1997;94(20) :10506-11.
-Based on the information in the passage, what effect would incorporation of dFTP in place of dTTP most likely have on the melting temperature of a DNA molecule of a fixed length?

Quizplus · Accepted Answer

11ecba2d_0ea2_133b_a3bf_473dd351390e_MD0008_00 The melting temperature T_m of DNA is the temperature at which 50% of the double helices in solution have been separated into single strands. It is determined by the strength of the intermolecular forces holding the strands together. An increased number of intermolecular interactions increases the T_m because more energy is required to disrupt them. Hydrogen bonding between bases (Watson-Crick hydrogen bonding) is one of the major intermolecular forces that hold DNA strands together. The number of hydrogen bonds between base pairs differs: G and C share three hydrogen bonds whereas A and T share only two. Therefore, DNA molecules with high GC content have higher melting temperatures than those with low GC content. The passage states that dFTP lacks Watson-Crick hydrogen bonding. Therefore, dFTP incorporation in place of dTTP will weaken interactions with adenine. Because there are fewer hydrogen bonds holding the strands together, less energy will be required to separate them. Consequently, DNA that incorporated dFTP instead of dTTP will most likely have a decreased melting temperature. (Choice A) The melting temperature is expected to decrease. Reduced base stacking would most likely decrease, not increase, T_m due to decreased hydrophobic interactions. (Choice C) Although dFTP is similar in shape to dTTP and specifically pairs with adenine, its inability to form hydrogen bonds with adenine makes the interaction weaker and results in a decreased melting temperature. (Choice D) The melting temperature of a DNA molecule is affected by the number of nucleotides. DNA molecules with more nucleotides have more intermolecular interactions (eg, hydrogen bonds) and increased melting points. However, the question states that the DNA molecule has a fixed length, so the number of nucleotides is not a factor in this scenario. Educational objective: The strands of a DNA double helix are held together by intermolecular interactions that include hydrogen bonds. Reducing the number of hydrogen bonds decreases the melting point of a double helix.

Passage DNA Polymerization Is One of the Most Conserved Mechanisms of of Genome