1. DNA Replication Part 2 Enzymology

2. Figure 11.10 The Polymerization Reaction

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

7. Sliding Clamps E. Coli dimer clampHuman 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 5 3 5 3 3 5 5 Helicase Topoisomerase ssDNA-binding proteins DNA polymerase RNA primer Leading strand Lagging strand RNA primer Primase

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'PO 4

12. Termination of Replication: Circular Chromosomes

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

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

15. 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 Telomere Sequence & Structure

17. 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

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

19. Telomeres Protect Ends of Chromosomes