DNA: Estructura , replicacion , transcripcion , procesamiento y mutaciones

1. DNA: Estructura, replicacion, transcripcion, procesamiento y mutaciones José A. Cardé- Serrano, PhDUniversidad Adventista de las Antillas Biol 223 - Genética Agosto 2011

2. Genome Size and Developmental Complexity

3. Chromatin Composition

4. Histone Proteins • Structural role in chromatin • Present in amounts equivalent to amounts of DNA • Major histone types: H1, H2a, H2b, H3, and H4 • Basic proteins • Arginine and Lysine are Abundant • Highly conserved proteins

5. Levels of DNA Packaging • 2-nm double-stranded DNA molecule • 11-nm nucleosomes • 30 nm chromatin fiber • Organization around a central scaffold

7. Nucleosomes

8. The 30 nm Fiber

9. DNA Around a Scaffold of Nonhistone Proteins

10. Human Metaphase Chromosomes

11. Centromeres • Constricted region of the chromosome • Necessary for proper segregation of chromosomes in mitosis and meiosis

12. Telomeres • Functions of telomeres • Protect the ends of linear DNA molecules from deoxyribonucleases • Prevent fusion of chromosomes ends or terminals • Facilitate complete replication of the ends of linear DNA molecules • Most telomeres contain repetitive sequences and a distinct structure.

13. Telomere Structure

14. 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?

15. 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?

16. Possible Modes of DNA Replication 3 Hipótesis posibles

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

18. The Origin of Replication in E. coli

19. Visualization of Replication in E. coli

20. Replication in E. coli

21. Replication is Bidirectional

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

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

24. DNA Polymerase I

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

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

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

28. 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

29. Proofreading

30. 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.

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. The E. coli Replisome

39. Ori C • Burbuja de replicacion • DNA A protein se pegan al repeat del core de 9 bb, se pegan entre 20 y 40 DNA A • se separan las cadenas se forma la burbuja • DNA B helicasa y DNA C se unen para formar el fork bidireccioinal • DNA T tambien de funcion desconcocida • Otras entre ellas la DNA girasa y las SSB • DNA primasa = RNA pol q hace primers (cadena leading un solo primer) • lagging primosoma = DNA primasa y DNA Helicasa • DNA pol III extiende • DNA topo provee los ejes de rotacion haciendo los nicks • SSB mantienen • DNA pol I reemplaza los primers de RNA • DNA ligasa unes los fragmentos de ocka • DNA girasa introdue el negative supercoil (DNA topoi)

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

41. Bidirectional Replication from Multiple Origins in Eukaryotes

43. The Eukaryotic Replisome

44. 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

45. Disassembly and Assembly of Nucleosomes

46. The Telomere Problem

47. Telomerase

48. Telomere Length and Aging • Most human somatic cells lack telomerase activity. • Shorter telomeres are associated with cellular senescence and death. • Diseases causing premature aging are associated with short telomeres.

49. The Central Dogma

50. Transcription and Translation in Prokaryotes • The primary transcript is equivalent to the mRNA molecule. • The mRNA codons on the mRNA are translated into an amino acid sequence by the ribosomes.