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Dna replication lec (2)

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process of DNA replication to understand medical genetics

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Dna replication lec (2)

  1. 1. 25-Jul-15 1
  2. 2. 25-Jul-15 2
  3. 3. 25-Jul-15 3 Dr Farah Deeba Khan www.wiley.com/college/pratt/.../animations/dna_replication/index.html
  4. 4. Learning Objectives • To understand the process of DNA replication. • To differentiate between Prokaryotic & Eukaryotic DNA replication. • To determine its importance. • To learn DNA repair mechanism. 25-Jul-15 4
  5. 5. 25-Jul-15 5 DNA Replication Transcription Translation
  6. 6. Replication The process of making an identical copy of a section of double-stranded DNA, using existing DNA as a template for the synthesis of new strands. 25-Jul-15 6 DNA Replication
  7. 7. Learning Outcomes Learning Outcomes Students should be able to answer………….. 25-Jul-15 7
  8. 8. 25-Jul-15 8 Learning Outcomes: Students should be able to answer…………..
  9. 9. 25-Jul-15 9
  10. 10. Why replicate DNA????? • If DNA never replicated, • mitosis would slowly halve the size of the genome until each cell would die. 25-Jul-15 10
  11. 11. CELL CYCLE Stage Activity Duration G1 Growth & increase in cell size 10 hr S DNA synthesis 8 hr G2 Post-DNA synthesis 5 hr M Mitosis 1 hr 25-Jul-15 11 When DNA replicates?
  12. 12. How Replication Works???? 25-Jul-15 12
  13. 13. 25-Jul-15 13 Models of Replication one each of the old and new strands Sections of the old and new pieces duplex dispersed randomly Old duplex is conserved with completely new strand
  14. 14. Requirements • DNA template • RNA Primer (Free 3' -OH group ) • dNTP (dATP, dGTP, dCTP, dTTP) • Proteins & Enzymes of DNA Replication – DNA Helicases – DNA single-stranded binding proteins (SSB-protein) – DNA Gyrase /topoisomerase – Primase – DNA Polymerase – DNA Ligase 25-Jul-15 14
  15. 15. Sequential Actions Initiation: • Recognition of the site of start (short sequence rich in AT base pairs) • Unwinding & Separation of dsDNA • Primer synthesis Elongation: • Add dNTPs to the existing strand • Form phosphodiester bonds • Correct the mismatch bases • Extend the DNA strand Termination: • Stop the replication 25-Jul-15 15
  16. 16. 25-Jul-15 16
  17. 17. I. Initiation 1. DnaA protein binding with AT rich regions in DNA  melting 25-Jul-15 17
  18. 18. 25-Jul-15 18 Initiation replicon.
  19. 19. Initiation….. 25-Jul-15 19 2. Helicases: bind to dsDNA and stimulate separation of the two strands.
  20. 20. 25-Jul-15 20 • Separation of dsDNA replication forks • Two replication forks move in opposite directions bidirectional replicon.
  21. 21. 25-Jul-15 21 3. Single-stranded binding proteins: bind, stabilize and keep the ssDNA separated. Initiation…..
  22. 22. 25-Jul-15 22 4. DNA Gyrase /Topoisomerase: prevents formation of supercoils
  23. 23. 25-Jul-15 23
  24. 24. Topoisomerase Type I • negative supercoils in bacteria • negative & positive in eukaryotes • ATP not required Type II • requires ATP • for interlocked supercoils after Chromosomal Replication (Prokaryotes & eukaryotes) 25-Jul-15 24
  25. 25. 25-Jul-15 25 5. RNA Primer: • strands of short ~ 10 bp, double stranded regions • have RNA base pairs with a free –OH on 3’-end • serve as starting point for DNA synthesis
  26. 26. 25-Jul-15 26
  27. 27. 25-Jul-15 27 6. Primase: RNA Polymerase adds (ATP, UTP, CTP, GTP) one at a time to 5’3’, anti-parallel to parent DNA strand
  28. 28. 25-Jul-15 28
  29. 29. 25-Jul-15 29 II. Elongation
  30. 30. 25-Jul-15 30 Direction of replication 5’3’ Elongation…
  31. 31. 7. DNA Polymerase III: adds (dATP, dTTP, dCTP, dGTP) one at a time to RNA primer, 5’3’, antiparallel to parent DNA strand 25-Jul-15 31 Elongation….
  32. 32. DNA Polymerase Type III • read in the 3' → 5' direction, • adds dNTPs at rate ~800 dNTPs/S • Pol III discriminate between correctly paired bases and incorrectly paired bases proofreading 25-Jul-15 32 Elongation…..
  33. 33. 25-Jul-15 33
  34. 34. 25-Jul-15 34
  35. 35. 25-Jul-15 35 III. Termination: •The RNA primers degraded by RNAse H & DNA Polymerase I (exonuclease), •gaps-filled with deoxyribonucleotides •sealed by the enzyme ligase.
  36. 36. 25-Jul-15 36
  37. 37. 25-Jul-15 37
  38. 38. 25-Jul-15 38
  39. 39. 25-Jul-15 39
  40. 40. 25-Jul-15 40 Termination…. In prokaryotes two replication forks meet at one point Ter
  41. 41. 25-Jul-15 41 In linear eukaryotic chromosomes, •DNA replicated until meets another origin of replication •Termination same
  42. 42. 25-Jul-15 42 •non-coding part of DNA at the ends of Ch…. •composed of several thousands repeats of hexameric sequence AGGGTT •Problem occurs at end of a linear chromosome is reached. •In this gap DNA is extended by telomerase. •This extension is called a telomere
  43. 43. 25-Jul-15 43 Mechanism of Action of Telomerase
  44. 44. Functions of telomeres • Prevent the termini from entangling & sticking • structural integrity • complete replication of chromosomes. • functional organization of chromosomes within the nucleus. • regulation of gene expression. • Serves as a molecular clock that controls the replicative capacity of human cells and their entry into senescence. 25-Jul-15 44
  45. 45. • Most human somatic cells lack telomerase activity but present in over 90% of cancerous and in vitro immortalized cells. • Shorter telomeres are associated with cellular senescence and death. • Diseases causing premature aging are associated with short telomeres. 25-Jul-15 45
  46. 46. 25-Jul-15 46

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