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DNA Synthesis: Replication Mechanism and Fidelity

This chapter explores the process of DNA replication, including the requirements for DNA polymerization and the mechanisms that ensure fidelity. It also discusses the structure and function of DNA polymerase enzymes.

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DNA Synthesis: Replication Mechanism and Fidelity

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  1. Chapter 2: DNA Synthesis (Replication) Required reading: Stryer’s Biochemistry 5th edition p. 127-128, 750-754, 759-766, 768-773 (or Stryer’s Biochemistry 4th edition p. 88-93, 799-809, 982-986, 809-814)

  2. DNA Replication is semi-conservative Meselson, Stahl 1958 • Both strands serve as templates for DNA synthesis • Each DNA molecule contains one • strand from original DNA and one • new strand

  3. DNA Polymerization Reaction Requirements for DNA polymerization • Template DNA (single stranded or double-stranded with a “nick”) • A primer strand with a free 3'- hydroxyl group (usually RNA) • Deoxynucleoside 5'-triphosphates (dATP, dGTP, dTTP and dCTP) • Mg2+ to activate the dNTPs • Polymerase and other accessory enzymes General reaction: 2Pi

  4. DNA Polymerization Reaction

  5. DNA Synthesis: addition of new dNTPsfollows Watson-Crick rules O H N N 2 NH N N N N NH 2 O G•C N NH 2 O N N HN N N O A•T Template base Incoming base G C C G T A A T

  6. 3’ 5’ Exonuclease

  7. 5’  3’ -exonuclease

  8. E. coli DNA Polymerase I Klenow Fragment 5' 3' Nucl. Polymerase N 3' 5‘ Nucl. C

  9. Typical Polymerase Structure: E. Coli Pol I fingers thumb palm polymerase exonuclease

  10. Polymerase with bound DNA

  11. Mechanism of phosphoryl transfer

  12. Polymerase fidelity mechanisms • Watson-Crick base pairing between the incoming dNTP • and the corresponding base in the template strand. • 2. H-bond formation between the minor groove of the new base pair • and the amino acids in the polymerase active site. • 3. Proofreading mechanism via 3' exonuclease that excises • incorrectly added nucleotides.

  13. 1. Correct Watson-Crick base pairing between the incoming dNTP and the corresponding base in the template strand induces conformational change required for polymerization reaction: Thumb Fingers

  14. 2. H-bond formation between the minor groove of the new base pair and amino acids in the polymerase active site:

  15. All Watson-Crick base pairs contain two H-bond acceptors at the same sites of the minor groove O N H N NH2 2 2 NH N N N N NH NH 2 2 O N NH 2 O N N HN N N HN 2 N O O N A•T N HN N N O G•C O N N NH N N O N T:A C:G

  16. 3. 3’-Exonuclease Proofreading function of DNA polymerases excises incorrectly added nucleotides.

  17. Fidelity of DNA Polymerization: Absolutely Essential!! Error Probability = Polymerization error (10-4) X 3' 5' Nuclease error (10-3) = 10-7 (1 in 10,000,000 nt)

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