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). dFTP is a nonpolar analog of dTTP that lacks Watson-Crick hydrogen bonding. Klenow fragment polymerase (KF), which has 3′-5′ but not 5′-3′ exonuclease activity, was incubated with a mixture of DNA template, primers, and dNTPs, including dFTP. 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 enzymeAdapted 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. Question 44The Klenow fragment used in the experiment would be able to perform which of the following repair processes?A.Correction of mismatched nucleotides in the middle of a completed strandB.Replacement of nucleotides at the 3′ end of the growing strandC.Excision of thymine dimers at the 5′ end of the growing strandD.Removal of damaged bases from the middle of the template strand
Question
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). dFTP is a nonpolar analog of dTTP that lacks Watson-Crick hydrogen bonding. Klenow fragment polymerase (KF), which has 3′-5′ but not 5′-3′ exonuclease activity, was incubated with a mixture of DNA template, primers, and dNTPs, including dFTP. 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 enzymeAdapted 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. Question 44The Klenow fragment used in the experiment would be able to perform which of the following repair processes?A.Correction of mismatched nucleotides in the middle of a completed strandB.Replacement of nucleotides at the 3′ end of the growing strandC.Excision of thymine dimers at the 5′ end of the growing strandD.Removal of damaged bases from the middle of the template strand
Solution
The Klenow fragment (KF) used in the experiment would be able to perform option B: Replacement of nucleotides at the 3′ end of the growing strand.
The Klenow fragment is a large protein fragment produced when DNA Polymerase I from E. coli is cleaved by the protease subtilisin. It has 5'-3' polymerase activity and 3'-5' exonuclease activity, but lacks 5'-3' exonuclease activity. This means it can add nucleotides to the 3' end of a primer or growing DNA strand and remove nucleotides from the 3' end if they are mismatched (proofreading), but it cannot remove nucleotides from the 5' end or in the middle of a DNA strand. Therefore, it would not be able to perform options A, C, or D.
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DNA replication is a highly coordinated process that involves a number of different enzymes. It is essential that the process is done correctly; otherwise, mutations will arise.DNA Replication by Madprime (CC0)Which of the following statements correctly pairs the enzyme involved in DNA replication with its function?Elimination ToolSelect one answerATopoisomerase: places down primers.BRNA polymerase: creates the new strands of DNA.CHelicase: breaks bonds between nucleotides.DPrimase: releases tension in the sugar-phosphate backbone.
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