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DNA REPLICATION WC: DNA replication is semi-conservative strands melt: form templates for copy

DNA REPLICATION WC: DNA replication is semi-conservative strands melt: form templates for copy Copy is reverse & complement of template Copy of other strand. Meselson & Stahl proved DNA replication is semi-conservative grew E. coli on 15 N to tell new DNA from old

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DNA REPLICATION WC: DNA replication is semi-conservative strands melt: form templates for copy

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  1. DNA REPLICATION WC: DNA replication is semi-conservative strands melt: form templates for copy Copy is reverse & complement of template Copy of other strand

  2. Meselson & Stahl • proved DNA replication is semi-conservative • grew E. coli on 15N to tell new DNA from old • make dense DNA

  3. Meselson & Stahl • 1) grew E. coli on 15N to tell new DNA from old • make dense DNA • 2) Xfer to 14N

  4. Meselson & Stahl • 1) grew E. coli on 15N to tell new DNA from old • make dense DNA • 2) Xfer to 14N • new DNA is light

  5. Meselson & Stahl • 1) grew E.coli on 15N to • tell new DNA from old • make dense DNA • 2) Xfer to 14N • new DNA is light • 3) measure r of F1 & F2 DNA by • centrifuging in CsCl

  6. Meselson & Stahl • measurerof F1 & F2 DNA • by centrifuging in CsCl • formsrgradients when spun • @500,000 x g

  7. Meselson & Stahl • measurerof F1 & F2 DNA • by centrifuging in CsCl • formsrgradients when spun • @500,000 x g • DNA bands where • ris same as CsCl

  8. Meselson & Stahl • Results • F0 DNA formsHHbandcontrolParental

  9. Meselson & Stahl • F0 DNA formsHH band • F1 DNA forms one higher band • Control Parental F1

  10. Meselson & Stahl • F0 DNA formsHH band • F1 DNA forms one higher band: HL • Control Parental F1

  11. Meselson & Stahl F0 DNA formsHH band F1 DNA forms one higher band: HL F2 DNA forms 2 bands: #1 same as F1 #2 same as 14N DNA Control Parental F1 F2

  12. Meselson & Stahl F2 DNA forms 2 bands: # 1 same as F1 #2 same as 14N DNA # 1 =HL # 2 = LL : DNA replication is semiconservative

  13. DNA replication 1) Replication begins atorigins of replication

  14. DNA replication 1) Replication begins atorigins of replication Polymerases are dumb!

  15. DNA replication 1) Replication begins atorigins of replication DNA polymerases are dumb! other proteins tell where to start

  16. DNA replication 1) where to begin? 2) “melting” DNA

  17. DNA replication • 1) where to begin? • 2) “melting” DNA • must melt DNA @ physiological T

  18. DNA replication must melt DNA @ physiological T Helicase melts DNA

  19. DNA replication must melt DNA @ physiological T Helicase melts DNA Forms “replication bubble”

  20. DNA replication • helicase melts DNA • Forms “replication bubble” • SSB proteins separate strands until they are copied

  21. DNA replication helicase melts DNA unwinding DNA increases supercoiling elsewhere

  22. DNA replication helicase melts DNA unwinding DNA increases supercoiling elsewhere DNA gyrase relieves supercoiling DNA gyrase

  23. DNA replication • 1) where to begin? • 2) “melting” • 3) “priming” • DNA polymerase can only add

  24. DNA replication • “priming” • DNA polymerase can only add • primase makes short RNA primers

  25. DNA replication • “priming” • primase makes shortRNAprimers • DNA polymerase adds to primer

  26. DNA replication • “priming” • primase makes short RNA primers • DNA polymerase adds to primer • later replace primers with DNA

  27. DNA replication • 1) where to begin? • 2) “melting” • 3) “priming” • 4) DNA replication

  28. DNA replication • 4) add bases bonding 5’ P to • 3’ OH @ growing end

  29. DNA replication 4) add bases bonding 5’ P to 3’ OH @ growing end Template holds next base until make bond

  30. DNA replication • Template holds • next base until • make bond • - only correct • base fits

  31. DNA replication • Template holds • next base until • make bond • - only correct • base fits • - energy comes • from 2 PO4

  32. DNA replication • energy comes from 2 PO4 • "Sliding clamp" keeps polymerase from falling off

  33. DNA replication • energy comes from 2 PO4 • "Sliding clamp" keeps polymerase from falling off • Proof-reading: only correct DNA can exit

  34. DNA replication • Proof-reading: only correct DNA can exit • Remove bad bases & try again

  35. DNA replication • Only make DNA • 5’ -> 3’

  36. Leading and Lagging Strands Only make DNA 5’ -> 3’ strands go both ways!

  37. Leading and Lagging Strands Only make DNA 5’ -> 3’ strands go both ways! Make leading strandcontinuously

  38. Leading and Lagging Strands Make leading strandcontinuously Make lagging strand opposite way

  39. Leading and Lagging Strands Make leading strandcontinuously Make lagging strand opposite way wait for DNA to melt, then make Okazaki fragments

  40. Leading and Lagging Strands Make lagging strand opposite way wait for DNA to melt, then make Okazaki fragments each Okazaki fragment has its own primer made discontinuously

  41. Leading and Lagging Strands each Okazaki fragment has its own primer made discontinuously DNA replication is semidiscontinuous

  42. Leading and Lagging Strands each Okazaki fragment has its own primer made discontinuously DNA replication is semidiscontinuous Okazaki fragments grow until hit one in front

  43. Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled

  44. Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled DNA ligase joins fragments

  45. Okazaki fragments grow until hit one in front RNAse H removes primer & gap is filled DNA ligase joins fragments Energy comes from ATP-> AMP

  46. DNA replication Real process is far more complicated! Proteins replicatingbothstrands are in replisome

  47. DNA replication Real process is far more complicated! Proteins replicatingbothstrands are in replisome: feed DNA through it

  48. DNA replication Proteins replicatingbothstrands are in replisome: feed DNA through it lagging strand loops out so make both strands in same direction

  49. lagging strand loops out so make both strands in same direction DNA pol detaches when hits previous primer, reattaches at next primer

  50. Bacterial DNA has one origin Euk have ARS ~ every 100,000 bp - speed DNA replication

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