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4 주차 수업자료

4 주차 수업자료. Chapter 10. Cell Division and DNA Replication. DNA replication occurs at replication fork. Replication fork Replisome: assembly of proteins responsible for DNA replication DNA replication is semi-conservative. Supercoiling: problem for replication. Circular chromosome

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4 주차 수업자료

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  1. 4주차 수업자료

  2. Chapter 10. Cell Division and DNA Replication

  3. DNA replication occurs at replication fork • Replication fork • Replisome: assembly of proteins responsible for DNA replication • DNA replication is semi-conservative

  4. Supercoiling: problem for replication • Circular chromosome • Bidirectional or theta replication • During replication, positive supercoils ahead of replisome (at replication forks) are removed by DNA gyrase • Inhibitor of gyrase: quinolone antibiotics

  5. DNA strand separation before DNA synthesis • DNA helicase: double helix DNA unwinding enzyme • SSB protein: prevents re-annealing of separate DNA strand

  6. DNA polymerase • 5’  3’ DNA synthesis • Leading strand (continuous) vs. lagging strand (discontinuous) • RNA primer (made by primase, DnaG) is required for new strand synthesis • Okazaki fragment: DNA fragments in lagging strand (1,000-2,000 bases)

  7. Nucleotide polymerization • 5’-P  3’-OH • Phospodiester linkage

  8. Precursors for DNA synthesis • ATP  deoxy ADP  dATP: by ribonucleotide reductase & kinase • UDP  deoxy UDP  dUMP  dTMP  dTDP  dTTP: by ribonucleotide reductase, phosphatase, thymidylate synthase, kinase & methyl group from THF • DHF  THF: by dihydrofolate reductase

  9. DNA polymerase III - replication assembly • Sliding clamp: two semicircular subunits of DnaN protein • Clamp-loading complex: load sliding complex onto DNA • Sliding clamp holds the core polymerase onto DNA • Core: DnaE (α-subunit for polymerization), DnaQ (ε-subunit for proofreading), HolE (θ subunit), Tau (τ-subunit for dimer maintenance)

  10. DNA polymerase III - proofreading • Kinetic proofreading activity by DnaQ (ε-subunit) • 3’  5’ exonuclease activity • Mismatch repair system

  11. Primosome for discontinuous DNA synthesis • Primosome: priming complex (priA + primase, DnaG) found in lagging strand

  12. Gap filling between Okazaki fragments • DNA polymerase I (RNaseH activity + polymerase activity + kinetic proofreading activity) • DNA ligase: seals nick • PolI used for nick translation

  13. Bacterial origin of replication: oriC • Origin of replication: DNA replication starts • Initiation complex: DnaA (binds to oriC to initiate replication), DnaB (helicase), DnaC (load DnaB onto DNA), gyrase, SSB

  14. Control of replication initiation by DNA methylation or membrane attachment • Dam methylase • Hemi-methylated DNA • right after replication • at replication origin, DnaA protein level drops  slow re-methylation • cannot be used to initiate replication • binds to cell membrane by SeqA (sequestration protein)

  15. Bacterial termination of replication: terC • Ter site • prevent movement of replication forks • binding of Tus protein, which prevent movement of DnaB helicase • not necessary

  16. Disentangling required after replication • Catenane: topoiosomerase IV can decatenate • TopoIV also works behind the replication fork • Crossover resolvase: required when recombination occurred

  17. Cell division after replication • Cell division by binary fission or splitting • Septum formation and cell elongation

  18. Generation time in bacteria • 20 min – several hours • Chromosome duplication: 40 min • Cell division: 20 min • If generation time is too short, bacteria could contain incomplete copies of chromosomes

  19. Replicon • Contains origin of replication  self replicate • In prokaryotes, ccDNA (protected from exonuclease • Example: plasmid

  20. How about the replication of linear DNA in eukaryotes? • Eukaryotic DNA: linear • Loss of 5’-end DNA after RNA primer is removed? • Telomere! • Protein primer instead of RNA primer

  21. Role of telomerase • Telomere (6-base repeat) shortening after each replication cycle • Telomerase: elongate 3’-end by adding several 6-bp repeat  fill in complementary strand by RNA primer, DNA polymerase, and ligase

  22. DNA replication in eukaryotes • Multiple origins in eukaryotic chromosome • Bi-directional replication (like bacteria) • Replication bubble forms • Pre-replicative complex • Pre-loading complex

  23. DNA replication in eukaryotes • DNA synthesis • semi-conservative replication • Helicase: MCM • polα iDNA • RFC  polδ/ε + sliding clamp (PCNA)

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