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DNA Replication and Recombination

Lecture 4. DNA Replication 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 and Recombination

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  1. Lecture 4 • DNA Replication 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 DNA primase

  16. DNA Replication at the Lagging strand RNA primer is 10 nucleotides long

  17. DNA Ligase DNA ligase joins the 3 end of the new DNA fragment to the 5 end of the previous fragment This enzymes seals a broken phosphodiester bond DNA ligase uses ATP to activate the 5 end at the nick

  18. DNA Helicase DNA double helix are tightly coupled. High temperature is needed to break them (95oC) Different Helicase on leading and lagging strand

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

  20. SSB Proteins DNA Covers total of 8 nucleotides, DNA bases remain exposed DNA complex

  21. PCNA

  22. Cycle of DNA Polymerase/Clamping Protein loading and unloading At the lagging strand

  23. Protein machinery for DNA replication

  24. A Moving Replication

  25. Structure of the Moving Complex

  26. DNA Topoisomerase prevent DNA tangling Reversible nucleases binds covalently to DNA backbone phosphate Breaking pdiester bond Reversible and reforms

  27. DNA topoisomerase I

  28. DNA topoisomerase II

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

  30. 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

  31. DNA Replication in Chromosome

  32. 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

  33. 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

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

  35. 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

  36. Addition of new histones Chromatin assembly factors (CAFs) help to add and assemble new nucleosomes Newly H3 and H4 are acetylated on their N-terminal tails after incorporated into chromatin AG are removed enzymatically

  37. Telomerase Structure Reverse transcriptase with RNA template to bind to DNA strands

  38. Telomerase and its function

  39. Summary • Specific DNA sequence determine replication origin, recruiting proteins to form replication machinery. relatively complex in eucaryotes • Bacteria has single replication origin. Eucaryotes have multiple origins and less defined. • Replication forks are activated at different times in eucaryotes • Telomere and telomerase

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