DNA Replication Ch 20 and 21

1. DNA ReplicationCh 20 and 21 • Review session tomorrow at 5 pm and Monday at 5 pm • Office hours by appointment next week • DNA replication

2. Meselson and Stahl

4. Semidiscontinuous vs. Continuous vs. Discontinuous

5. Semidiscontinuous Replication • Leading strand • Replicated continuously • Lagging strand • Discontinuous replication • Okazaki fragments • Ligase seals nicks

6. Priming

7. Primers • RNA • 10-12 nt long

8. Direction of Replication • Most eukaryotic and prokaryotic organisms replicate bidirectionally (q mode) • Origin of replication • Two replication forks • Some plasmids replicate unidirectionally (ColE1) • Rolling circle • fX174 circular ssDNA • l phage dsDNA

9. fX174 circular ssDNA

10. l Phage ds DNA

11. Enzymology of Replication • >30 proteins • Strand separation • DNA helicase • DnaB • ATPase • Stimulated by the primase DnaG

12. Single-Strand DNA-Binding proteins (SSB's) • Bind cooperatively and stabilize ssDNA • Aids helicase and protects DNA from from degredation • Can unwind short hairpins • Unidentified in eukaryotes • RF-A in SV40 • Stimulate homologous DNA polymerases

14. Topoisomerases • Introduce single (type I) or double (type II) stranded breaks in the DNA • Allows DNA to “swivel” to prevent strain caused by the unwinding of the helix by helicase • Underwinding • supercoils • DNA gyrase (type II) • E. coli type I topoisomerases can only relax negative supercoils • Eukaryotes and Archaea can use type I topoisomerases to relax positive supercoils

16. Topoisomerase II • Uses ATP to break and reform phosphodiester bonds • Forms a covalently bonded intermediate wherein DNA is bound to the enzyme, conserving the energy of the phosphodiester bond • Have 2 “jaws” • G-segment binds one segment of DNA and contains the gate through which the other segment will pass • T-segment binds the other DNA segment that will be transported through the gate

17. DNA Polymerases in E. coli • Three polymerases • Pol I • First to be discovered (Kornberg) • 5'-3' polymerase activity • 3'-5' exonuclease activity proofreads newly synthesized DNA • 5'-3' exonuclease activity degrades a strand ahead of the advancing polymerase so that it can remove and replace the strand • DNA repair

18. Mild proteolytic treatment yields the Klenow fragment with polymerase activity and 3'-5' proofreading and a smaller fragment with 5'-3' exonuclease activity • Ligase seals nicks

19. Pol II: not required for DNA replication • Pol III: primary DNA polymerase • Pol III holoenzyme has 10 peptides • Core a, e, q • a contains polymerase activity • e Contains 3'-5' exonuclease activity • q Role unknown

20. Eukaryotic DNA polymerases • Pol a priming of replication strands • Pol d elongation of both strands • Pol b DNA repair • Pol e DNA repair • Pol g replication of mitochondrial DNA

21. Processivity • Pol d processivity is enhanced by PCNA (proliferating cell nuclear antigen) • The other nuclear polymerases are not very processive

22. E. coli Replication Initiation • Initiation • OriC • Binds DnaA and ATP with the HU protein forming a multimer destabilizing the adjacent DNA region • DnaB binds to the melted DNA • DnaC binds to DnaB and helps it bind the DNA • Primase binds to this pre-priming complex and converts it to the primosome

23. Eukaryotic Replication Initiation • Autonomously replicating sequences (ARS's)

24. Elongation in E. coli • Pol II core requires b subunit which forms a dimer that is ring-shaped • The b clamp interacts with the a subunit and holds the core to the DNA • The clamp loader (known as the g complex) loads b onto the DNA • The g complex consists of g, d, d', c, and y • The clamp loader does not remain associated during replication • Clamp loading is ATP dependent

28. Bacterial Termination • Decatnation • Tus proteins bind Ter sites • This arrests the forks • Unreplicated section is denatured • Repair synthsis fills gaps • Topoisomerase IV decatenates

29. Eukaryotic Termination • Telomeres • Telomerase