Chapter 10 Replication of DNA and Chromosomes

1. Chapter 10Replication of DNA and Chromosomes José A. Cardé- Serrano, PhDUniversidad Adventista de las AntillasBiol 223 – Genética Agosto 2010

2. Chapter Outline • Basic Features of DNA Replication In Vivo • DNA Polymerases and DNA Synthesis In Vitro • The Complex Replication Apparatus • Unique Aspects of Eukaryotic Chromosome replication

3. Introducción • Replicación – procesos por el cual la célula genera una copia de su material genético usando como molde una previa • Iniciación  extensión  terminación • 30,000 bpm vs 3,000 bpm • 1 error / billón • Implicaciones en los gemelos?

4. DNA replication occurs semiconservatively, is initiated at unique origins, and usually proceeds bidirectionally from each origin of replication. Basic Features of DNA Replication In Vivo

5. DNA Replication is Semiconservative • Each strand serves as a template • Complementary base pairing determines the sequence of the new strand • Each strand of the parental helix is conserved in a hybrid new molecule • Problema: Pregunta? • Como se replicará el DNA?

6. Possible Modes of DNA Replication 3 Hipótesis posibles

7. The Meselson-Stahl Experiment:DNA Replication in E. coli is Semiconservative

8. Semiconservative Replication in Eukaryotes

9. Semiconservative Replication in Eukaryotes - Evidence

10. The Origin of Replication in E. coli

11. Visualization of Replication in E. coli

12. Replication in E. coli

13. Replication is Bidirectional

14. Replication is Bidirectional

15. Key Points • DNA replicates by a semiconservative mechanism: as the two complementary strands of a parental double helix unwind and separate, each serves as a template for the synthesis of a new complementary strand. • The hydrogen-bonding potentials of the bases in the template strands specify complementary base sequences in the nascent DNA strands. • Replication is initiated at unique origins and usually proceeds bidirectionally from each origin.

16. Much of what we know about DNA synthesis was deduced from in vitro studies. DNA Polymerases and DNA Synthesis In Vitro

17. Requirements of DNA Polymerases • Primer DNA with free 3'-OH • Template DNA to specify the sequence of the new strand • Substrates: dNTPs • Mg2+

18. DNA Polymerase I

19. DNA Polymerase I:5'3' Polymerase Activity

20. DNA Polymerase I:5'3'Exonuclease Activity

21. DNA Polymerase I:3'5' Exonuclease Activity

22. DNA Polymerases • Polymerases in E. coli • DNA Replication: DNA Polymerases III and I • DNA Repair: DNA Polymerases II, IV, and V • Polymerases in Eukaryotes • Replication of Nuclear DNA: Polymerase  and/or  • Replication of Mitochondrial DNA: Polymerase  • DNA Repair: Polymerases and • All of these enzymes synthesize DNA 5' to 3' and require a free 3'-OH at the end of a primer

23. DNA Polymerase III is the True DNA Replicase of E. coli

24. Proofreading

25. Key Points • DNA synthesis is catalyzed by enzymes called DNA polymerases. • All DNA polymerases require a primer strand, which is extended, and a template strand, which is copied. • All DNA polymerases have an absolute requirement for a free 3'-OH on the primer strand, and all DNA synthesis occurs in the 5' to 3' direction.

26. Key Points • The 3'5' exonuclease activities of DNA polymerases proofread nascent strands as they are synthesized, removing any mispaired nucleotides at the 3' termini of primer strands.

27. DNA replication is a complex process, requiring the concerted action of a large number of proteins. The Complex Replication Apparatus

29. DNA Replication • Synthesis of the leading strand is continuous. • Synthesis of the lagging strand is discontinuous. The new DNA is synthesized in short segments (Okazaki fragment) that are later joined together.

30. Okazaki Fragments Experiment • Crecer fagos y Ecoli en medio con 3H Timina por periodos cortos (pulso y seguimiento) • Aislar el DNA y centrifugarlos para medir su grado de Sedimentacion • A periodos cortos de 5, 10, 15, 20 segundos se obtienen fragmentos bien cortos • A periodos mas largos, la radioactividad se ve incluida en fragmentos mas grandes

31. DNA Ligase Covalently Closes Nicks in DNA

32. RNA Primers are Used to Initiate DNA Synthesis

34. DNA Helicase Unwinds the Parental Double Helix

35. Single-Strand DNA Binding (SSB) Protein

36. DNA Topoisomerase I Produces Single-Strand Breaks in DNA

37. The Replication Apparatus in E. coli

38. Prepriming at oriC in E. coli

39. The E. coli Replisome

40. Rolling-Circle Replication

41. Key Points • DNA replication is complex, requiring the participation of a large number of proteins. • DNA synthesis is continuous on the progeny strand that is being extended in the overall 5'3' direction, but is discontinuous on the strand growing in the overall 3'5' direction.

42. Key Points • New DNA chains are initiated by short RNA primers synthesized by DNA primase. • The enzymes and DNA-binding proteins involved in replication assembled into a replisome at each replication fork and act in concert as the fork moves along the parental DNA molecule.

43. Although the main features of DNA replication are the same in all organisms, some processes occur only in eukaryotes. Unique Aspects of Eukaryotic Chromosome Replication

44. DNA Replication in Eukaryotes • Shorter RNA primers and Okazaki fragments • DNA replication only during S phase • Multiple origins of replication • Nucleosomes • Telomeres

45. Bidirectional Replication from Multiple Origins in Eukaryotes

47. The Eukaryotic Replisome

48. Eukaryotic Replication Proteins • DNA polymerase -DNA primase—initiation; priming of Okazaki fragments • DNA polymerase —processive DNA synthesis • DNA polymerase —DNA replication and repair in vivo • PCNA (proliferating cell nuclear antigen)—sliding clamp • Replication factor-C Rf-C)—loading of PCNA • Ribonuclease H1 and Ribonuclease FEN-1—removal of RNA primers

49. Disassembly and Assembly of Nucleosomes