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DNA Replication, Repair, and Recombination

DNA Replication, Repair, and Recombination. DNA Maintenance. Mutation rate are extremely low 1 mutation out of 10 9 nucleotides per generation. DNA replication Separation, Base pair. The Chemistry of DNA replication. DNA Synthesis by DNA polymerase. DNA Polymerase

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DNA Replication, Repair, and Recombination

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  1. DNA Replication, Repair, and Recombination

  2. DNA Maintenance • Mutation rate are extremely low • 1 mutation out of 109 nucleotides per generation

  3. DNA replication Separation, Base pair

  4. The Chemistry of DNA replication

  5. DNA Synthesis by DNA polymerase

  6. DNA Polymerase Nucleotide polymerizing enzyme, first discovered in 1957

  7. DNA replication with two forks

  8. This Doesn’t Work!

  9. DNA replication Fork

  10. DNA Proofreading

  11. Structures of DNA polymerase during polymerizing and editing E: exonucleolytic; P: polymerization

  12. Why 5’->3’? The need for accuracy

  13. Site-directed mismatch repair in eucaryotes In procaryotes, old DNAs are usually methylated on A while newly synthesized ones are not. So Cells can distinguish old and newly synthesized DNAs and mutate mismatches on new ones.

  14. DNA ProofreadingRNA usually doesn’t have this. Why? • Pairing, correct nucleotide has higher affinity binding to the moving polymerase • Un-Paired nucleotide is easier to be off before covalent ligation, even after binding. • Exonucleotic proofreading • Strand directed mismatch repair

  15. DNA Primer synthesis On Lagging strand

  16. DNA Replication at the Lagging strand

  17. DNA Ligase

  18. DNA Helicase DNA double helix are tightly coupled. High temperature is needed to break them (95oC)

  19. DNA Binding Protein SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins

  20. SSB Proteins DNA

  21. DNA Clamping Protein

  22. Cycle of DNA Polymerase/Clamping Protein loading and unloading At the lagging strand (how about leading strand?)

  23. Protein machinery for DNA replication

  24. A Moving Replication

  25. Structure of the Moving Complex

  26. DNA winding

  27. DNA topoisomerase I

  28. DNA topoisomerase II

  29. DNA topoisomerase II

  30. Mammalian replication Fork (eucaryote, DNA polymerase (primase) a synthesize RNA/DNA, DNA polymerase delta is the real polymerase)

  31. Summary • DNA replication 5’->3’ • DNA proof reading • Lagging strand, back-stitching, Okazaki fragment • Proteins involved: • DNA polymerase, primase • DNA helicase and single-strand DNA-binding protein (SSB) • DNA ligase, and enzyme to degrade RNA • DNA topoisomerases

  32. DNA Replication in Chromosome

  33. DNA replication in Bacterial Genome

  34. Initiating Proteins for DNA replication 1. Initiator protein, 2. helicase binding to initiator protein, 3. helicase loading on DNA, 4. helicase opens the DNA and binds to primase, 5. RNA primer synthesis, 6. DNA polymerase binding and DNA synthesis

  35. Regulation for DNA replication In Bacteria, hemimethylated origins are resistant to initiation, delayed methylation leads to delayed initiation at the second phase Dam methylase

  36. DNA replication in eucaryotes Multiple replication origin 50 nucleotides/second, autoradiography

  37. The four standard phases of a eucaryotic cell DNA replication occurring at S Phase (DNA synthesis phase) G1 and G2, gap between S and M

  38. Different regions of a chromosome are replicated at different times Arrows point to the replicating regions at different times

  39. Some facts about Replication in eucaryotes • Multiple replication origins occurring inclusters (20-80) (replication units) • Replication units activated at different times • Within replication units, replication origins are separated 30,000-300,000 pairs apart. • Replication forks form in pairs and create a replication bubbles moving in opposite directions • Different regions on the same chromosome are replicated at distinct times in S phase • Condensed Chromatin replicates late, while less condensed regions replicate earlier

  40. The search and identification of DNA replication sequence ARS: autonomously replicating sequence Only histidine expression can help cells to survive

  41. The origins of DNA replication on chromosome III of the yeast S. cerevisiae

  42. A close look at an origin of replication in yeast ORC: origin recognition complex B1, B2, B3: other regions binding to required proteins

  43. The replication origins of human genes are more complex Even far distant DNA sequences could be important

  44. Histone remains associated with DNA In vitro experiments DNAs with different sizes are replicated. Only the daughter DNAs replicated from parental DNA with histones showed histone binding

  45. Addition of new histones Chromatin assembly factors (CAFs) help to add and assemble new nucleosomes

  46. Bacteria DNAs are circular, not a problem There is a problem for eucaryote DNAs: ??? Hint: Telomere

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