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DNA Replication

DNA Replication. Tsung-Luo Jinn. Three stages of DNA Replication. Initiation: primosome , a protein complex act of initiation. Elongation: replisome , a protein complex, associate with particular DNA structure to unwind the DNA

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DNA Replication

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  1. DNA Replication Tsung-Luo Jinn

  2. Three stages of DNA Replication Initiation: primosome, a protein complex act of initiation Elongation: replisome, a protein complex, associate with particular DNA structure to unwind the DNA and synthesis daughter strands Termination: Tus protein and ter termination site

  3. Conditional lethal mutants: Temperature-sensitive mutants --the dna genes Quick-stop mutants--the major class Defect in elongation Slow-stop mutants--the smaller class Defect in initiation

  4. Function of DNA polymerase --the enzyme that can synthesis a new strand on a template strand In E.Coli DNA polymerase I: coded by polA DNA polymerase II: coded by polB DNA polymerase III:coded by polC --multisubunit In Phage--T4 ,SPO1 ,T5 ,T7 Code for DNA polymerase

  5. II III DNA polymerase I polymerase 3‘ ＋ ＋ ＋ 5‘ exonuclease 5‘ ＋ ＋ ＋ 3‘ ＋ － － exonuclease 3‘ 5‘ － － ＋ Replicase MW (kDa) 90 900 103 Numbers 400 ??? 10-20 Bioactivity 1 0.05 15 Gene pol A pol B pol C＊ Summary In Bacteria

  6. In Eukaryotic β γ ε δ α DNA polymerase Nu Nu Mito Nu Nu location Priming replication replication function repairing repairing replicase 5‘-3’ plolymerization ＋ ＋ ＋ ＋ ＋ － － ＋ ＋ ＋ 3‘-5’ exonuclease

  7. Errors in DNA synthesis Substitution --misspairing Determined by proofreading Frameshifts --insertion, deletion Affected by processivity Nonsense --early stop

  8. The fidelity of DNA replication Control at two different stages: Control at the incoming base --presynthetic control Proofreading control * ~10-6

  9. polymerization --an 5‘ to 3’ elongation

  10. The all DNA polymerase in Bacteria with 3’-5’ exonuclease activity---proofreading DNA polymerase I (103kDa) -- proteolytic treatment C-large fragment (68kDa) with polymerase and 3‘-5’ exonuclease activity --Klenow fragment N-small fragment(35kDa) --5’-3’ exonuclease (up to ~10 bases at a time)

  11. The Nick translation DNA polymerase I --in Nicked DNA --in vitro Need: 5‘-3’ polymerase 5‘-3’ exonuclease 3‘-5’ exonuclease * Rolling circle

  12. Priming reaction in replication With different way Primase: RNA polymerase ~10 bases Nicked DNA 3‘-OH Terminal protein:Ser :dCMP-3’OH

  13. Right Hand structure of DNA polymerase A-form DNA B-form DNA

  14. DNA replication A semidiscontinuous replication Leading strand: a continuous strand Lagging strand: a series fragments are jointed ~10-20 kb length Okazaki fragments

  15. The semidiscontinuous replication discontinuous continuous

  16. Primase,DNA Polymerase III DNA Polymerase I, DNA Ligase Primase:Code by dnaG --RNA polymerase --11-12 bases primer for DNA synthesis --primer start with the sequence pppAG, in 3’-GTC-5’ in template DNA polymerase III DNA polymerase I DNA ligase * Mammalian system

  17. Ligation ligase Phage--ATP E.coli--NAD Nicotinamide adenine dinucleotide *T4 DNA ligase

  18. Conversion of ψX 174 ds DNA into ssDNA in vitro TraY: bind to the oriT TraI: A protein,a relaxase, bind to TraY in the oriT Rep: helicase--unwinding DNA --ATP dependent SSB:single-strand DNA binding protein helicase --Prevent DNA reform to ds DNA

  19. Two types of priming in E.Coli OriC and ψX systems OriC: the bacteria origin DnaB--helicase (5’-3’) ψX: the phage origin DnaB--helicase (5’-3’) require primosome see page398 to replace SSB

  20. Problem in simultaneously synthesizing leading and lagging strands

  21. DNA polymerase III subunits subunits assembly

  22. The dimer surround the duplex, providing the sliding clamp that allow the holoenzyme to slide along DNA

  23. The replication fork Coordinating synthesis of leading and lagging strands DnaB:helicase DnaG:primase τ lagging strands polymeaseIII leading strands Increase synthesis speed Prevent polymerase fall off

  24. Dissociation and Reassociation of βclamp during DNA replication

  25. Termination Tus protein and termination of replication at ter site In E.Coli Tus protein (36 kDa) Terwith 23 bases consensus sequences See page 354 Stop helicase to unwind DNA Countra-helicase activity

  26. Summary The protein component of replication complex

  27. Initiation of replication fork at replication origin In E.Coli Ori C origin Ori λ origin

  28. Initiation of replication Ori C in vitro --Kornberg, 1953 A minimal origin DnaA, DnaB, DnaC, HU, Gyrase, SSB--primosome ATP needed DnaG: primer--replication

  29. Immunolocalization of replication complex at Ori C origin-by antibody against DnaB The DnaB:DnaC complex ~480kDa --- ~6nm

  30. Initiation of replication Ori λ in vitro Replication is activated by genes O and P transcription—away from ori. O protein bind to ori to generate a spherical structure -- O-some(~11nm), and then interacted with P protein bind to the replication origin within geneO -- primosome--DNA bending O protein==DnaA P protein==DnaC DNA replication, is trigger after P protein release DnaB, Gyrase, SSB, Dnak,DnaJ DnaG: primer--replication

  31. How to ensure initiation of replication only once per cycle ?

  32. Methylated DNA in the origin, can be distinguished from the replicated DNA In OriC, 11 copies of GATC Adenine-N6-CH3 by Dam methylase, before replication Daughter strands with hemimethylated DNA, can not be used to initiated a replication cycle Delay remethylation in oriC---delay replication

  33. The methylated DNA

  34. The model The membrane bound inhibitor binds to hemimethylated DNA Remethylated DNA, inhibitor releases DnaA binds to oriC --initiate replication

  35. How to control the multiple replicons is activated only once time in a single cycle ?

  36. HOW !! By rate-limiting component which function only once at the origin--licensing factor Two purposes Prevent more than one cycle of replication--by removing the component Makes the replication initiation dependent on cell division

  37. The licensing factor in Yeast ORC: origin recognition complex, bind to A and B1 in ARS Cdc6, a highly unstable protein (half-life＜5 min), synthesis only in the G1 phase Cdc6, allow Mcm bind to complex Replication initiation--Cdc6-Mcm are displaced

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