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DNA Replication

DNA Replication . Part 2 Enzymology. The Polymerization Reaction. Figure 11.10. Main E. coli DNA Polymerases. DNA polymerase I Single subunit enzyme Degrades RNA primers and fills gaps DNA repair 5' 3' polymerase activity 3’5’ exonuclease activity – proofreading function

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DNA Replication

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  1. DNA Replication Part 2 Enzymology

  2. The Polymerization Reaction Figure 11.10

  3. Main E. coli DNA Polymerases • DNA polymerase I • Single subunit enzyme • Degrades RNA primers and fills gaps • DNA repair • 5'3' polymerase activity • 3’5’ exonuclease activity – proofreading function • 5’3’ exonuclease activity – primer removal & repair function • DNA polymerase III • Main DNA polymerase for replication of genome and plasmids • Multiple subunit holoenzyme – different subunits for pol and exo activities • 5'3' polymerase activity • 3’5’ exonuclease activity – proofreading function

  4. Eukaryotic DNA Polymerases

  5. Catalytic domain of E. coli DNA Polymerase I

  6. E. coli DNA Pol III Holoenzyme holoenzyme Pol III*

  7. Sliding Clamps E. Coli dimer clamp Human trimer clamp Subunit  PCNA

  8. Enzymology of DNA Synthesis • DNA-dependent DNA Polymerases • require a DNA template • Require a free 3'-OH • a primer • NTP added to 3'-OH of growing polynucleotide chain • 5'  3' synthesis

  9. Mechanics of DNA Synthesis

  10. Architecture of the Replisome Topoisomerase Leading strand Helicase 3¢ 3¢ 5¢ 5¢ 5¢ 3¢ 5¢ Primase Lagging strand RNA primer DNA polymerase RNA primer ssDNA-binding proteins

  11. DNA Pol I Finishes the Job • DNA Pol I has 5'3' exonuclease function that destroys RNA primer • As RNA is removed, DNA is replaced by the 5'3' polymerase function • DNA ligase seals the gap by joining the 3'OH to the 5'PO4

  12. Termination of Replication: Circular Chromosomes

  13. What happens at the end of a linear chromosome? • The need for a primer poses a problem at the 3’ end of linear chromosomes Figure 11.24

  14. Telomeres and DNA Replication • Linear eukaryotic chromosomes have telomeres at both ends • The term telomere refers to the complex of telomeric DNA sequences and bound proteins

  15. Telomere Sequence & Structure Figure 11.23 • Telomeric sequences consist of hexanucleotide or heptanucleotide repeats • The very end of telomeres are a 12-16 nucleotides 3’ overhang formed by removal of the RNA primer after DNA synthesis

  16. Replication of Telomere Sequences • In mammalian somatic cells, 100-1000 telomere repeats are lost with each round of replication • Telomere sequence must be maintained in gamete precursors • Done by telomerase • Telomerase • utilized a RNA template to extent the 3’ end of one strand at the telomere • telomerase has reverse transcriptase enzyme activity • The RNA is complementary to the DNA sequence found in the telomeric repeat

  17. Telomerase & Telomere Extension The complementary strand is made by primase, DNA polymerase and ligase Step 1 = Binding Step 2 = Polymerization Reverse transcriptase Step 3 = Translocation Figure 11.25 RNA primer

  18. Telomeres Protect Ends of Chromosomes

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