CHAPTER 8.pptx - CHAPTER 8 The Chemistry Of DNA Synthesis • DNA synthesis requires two key substrates deoxynucleoside triphosphates – dGTP dCTP dATP

CHAPTER 8.pptx - CHAPTER 8 The Chemistry Of DNA Synthesis...

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CHAPTER 8
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The Chemistry Of DNA Synthesis DNA synthesis requires two key substrates: deoxynucleoside triphosphates – dGTP, dCTP, dATP, dTTP; and a primer:template junction. DNA is synthesised by extending the 3’ end of the primer. A phosphodiester bond is formed in an S N 2 reaction. The hydroxyl group at the 3’ end of the primer strand attacks the α- phosphoryl group of the incoming nucleoside triphosphate.
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The Chemistry Of DNA Synthesis The leaving group of the reaction is pyrophosphate made up of the β and γ phosphates of the nucleotide substrate. The hydrolysis of the leaving pyrophosphate provides the driving force for DNA synthesis.
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The Mechanism of DNA Polymerase DNA polymerase uses a single site to catalyse DNA synthesis. It accomplishes this by exploiting the nearly identical geometry of the A:T and G:C base pairs. DNA polymerase monitors the ability of the incoming nucleotide to base pair correctly rather than monitoring the exact incoming nucleotide. DNA polymerase is able to distinguish between rNTPs and dNTPs, even though the concentration of rNTPs is tenfold that of dNTPs.
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The Structure of DNA Polymerase DNA polymerase resembles a partially closed right hand. Three domains based on the analogy with a right hand: thumb, fingers, and palm.
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DNA Polymerase: Domain functions The catalytic site is located at the palm domain. The palm domain is composed of a β sheet. The catalytic site bind two divalent metal ions. One of the metal ions reduces the affinity of the 3’-OH of the nucleotide for its hydrogen, generating a 3’O- that is primed for a nucleophilic attack of the α-phosphate of the incoming dNTP.
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DNA Polymerase: Domain functions The second metal ion coordinates the negative charges of the β and γ phosphate of the incoming dNTP and stabilises the pyrophosphate produced. The palm domain monitors the base pairing of the most recently added nucleotides by making extensive hydrogen bond contacts with base pairs in the minor groove of the newly synthesised DNA. These hydrogen bond contacts only form if the recently added nucleotides are correctly base paired.
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DNA Polymerase: Functions of Domain Once correct base pairing is established between the incoming nucleotide and the template, the finger domain moves to enclose the dNTP. This closed form of the polymerase hand stimulates catalysis by moving the incoming nucleotide in close contact with the catalytic metal ions. The finger domain also helps in presenting the appropriate template base to the catalytic site by bending the phosphodiester backbone between the first and second bases of the template.
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DNA Polymerase: Domain functions The thumb domain is not intimately involved in catalysis.
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  • Fall '19
  • DNA, CHEMISTRY OF DNA SYNTHESIS

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