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Replication

Replication. دانشگاه علوم پزشكي وخدمات بهداشتي درماني تهران. Dr. Parvin Pasalar Tehran University of Medical Sciences. Objectives: To know and explain What are needed for Replication Template properties Building Blocks Enzymes Protein Factors Different Stages of Replication

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Replication

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  1. Replication دانشگاه علوم پزشكي وخدمات بهداشتي درماني تهران Dr. Parvin Pasalar Tehran University of Medical Sciences

  2. Objectives: • To know and explain • What are needed for Replication • Template properties • Building Blocks • Enzymes • Protein Factors • Different Stages of Replication • Initiation • Elongation • Termination • Difference between Replication in eu and pro • Toxins and antibiotics that inhibit replication

  3. Replication( overall view) • During cell division Whole DNA must replicate • The DNA double helix unwinds • The exposed bases bind to free floating nucleotides in the nucleoplasm • DNA polymerase binds the complimentary nucleotides

  4. Replication in Prokaryotes

  5. What are needed for Replication • 1- Permission (licensing) • 2- Template ( Double Stranded DNA) • 3- Building blocks ( dNTPs & NTPs) • 4- Enzymes • 5- Energy ( Energy of dNTPs & ATP) • 6- Protein Factors

  6. The Permission

  7. The Permission:Methylated DNA in the origin, can be distinguished from the replicated DNA

  8. The template • It is a double stranded DNA • It should have a specific sequence (origin) for replication to be started • It is replicated in semiconservative manner • It is replicated bidirectionaly

  9. Replication / The origin • Replication starts from Origin • Origin has specific AT rich sequences • Because of the shape of the origin, replicating bacteria DNA is called teta form A minimal Origin sequence is consisted of : 4 of 9 mer 3 of 13 mer Ori Teta form 245bp

  10. Enzymes of Replication 1- DNAPs ( DNA synthesis, gap filling) 2- RNAP =primase ( priming DNA synthesis) 3- Helicases ( opening the helix turns) 4- Topoisomerase (removing the superheix turns) 5- Ligase ( sealing the nicks between Okazaki fragments)

  11. DNA Polymerases in Bacteria II III DNA polymerase I polymerase 3‘ ＋ ＋ ＋ 5‘ 5‘ ＋ ＋ ＋ 3‘ Exonuclease ( Proof reading) ＋ － － exonuclease 3‘ 5‘ 600 30 9000 TNO( nt/min) Mass (kDa) 90 900 103 Numbers/ cell ??? 10-20 400 Bioactivity 1 0.05 15 Gene pol A pol B pol C＊

  12. 2- Enzymes/b: Primase( Dna G) • DNAPs can not initiate DNA synthesis (no de novo synthesis) and there must be small pre-existing primers • The enzyme responsible for primer synthesis is called Primase • It synthesize the primer from 5’ to 3’ • It has not exonuclease activity • It is part of primosome

  13. 2- Enzymes/c: Topoisomerases • Enzymes which relieve stress on the DNA by allowing free rotation around a single or double stranded DNA • There are two classes: 1 & 2 • Class 2 in bacteria is calledgyrase. • Gyrasereduces two turn of DNA each time ( convert +ve superhelix into –ve)

  14. 2- Enzymes/d: Helicases Enzyme which catalyze the unwinding and separation ( bearking H-bonds) of the parental double helix by using ATP • There are different types of helicases : • Dna B • Other Helicases

  15. 2- Enzymes/e: Ligase Ligases seal nicks in DNA The energy is provided by ATP or NAD Phage--ATP E.coli--NAD Eukaryote--ATP *T4 DNA ligases can join two blunt DS DNA

  16. Protein Factors of Replication • Dna A direct primosome to the origin • Dna B( Helicase activity) • Dna C & C’ are co-activator for primase • Dna G( primase) • SSBP binds to each ss DNA, keep the separated strands apart • Tus recognize the ter sequence for termination of replication

  17. Stages in Replication in Prokaryotes

  18. Different Stages • Recognition of the Ori sequence by Dna A • Initiation by primosome • Elongation by replisome • Termination at ter sequence by tus protein

  19. Initiation of replication at Ori C

  20. Recognition & Initiation of replication 1- Recognition of the origin by Dna A molecules 2- Twisting of DNA around them result in a short unwinded DNA( loop) 3- Progressive unwinding of the DNA by Dna B and eye loop formation 4-Assembly of primosome at replication origin 5- Synthesis of the primer by primase followed by DNAP activity

  21. 2- Elongation: 5’ to 3’ PDE bond formation Replisome, a protein complex, associate with particular DNA structure to unwind the DNA and synthesis daughter strands. Pyrophosphate hydrolysis is necessary for irreversibility of this reaction

  22. Simultaneous synthesis of two strands of DNA The two strands are run in opposite direction The DNA synthesis is only from 5’ to 3’ The two strands are synthesized Simultaneously But how ? One strand is made continuously (leading strand) and The other is made discontinuously (lagging strand, Okazaki fragments)

  23. OKAZAKI FRAGMENT Its structure: A short sequence(10 nt) of RNA primer + about 100 nt of DNA How it is made: Primer by primase and DNA by DNAP III How it is joined to the other fragments: First the primer of the previous Okazaki fragment is hydrolyzed and replace by DNAP I, then the two ends of DNA is linked by ligase

  24. Discontinuous Replication Stages 1- Primer synthesis by primase 2- DNA synthesis by DNAP III 3- Removal of primer by DNAP I 4- DNA synthesis by DNAP I 5- Sealing the nick between two Okazaki fragments by ligase Nick translation: is referred to these last three stages Priming Extension Removal of primer Gap filling Nick ligation

  25. How the two strands are synthesize in same direction?

  26. 3- Termination of replication in E.Coli 1- Ter sequences is a consensussequences with 23 bases 2- Tus protein is a 36 kDa protein that binds to the ter site 3- Contra helicase activity of the Ter- Tus complex 4- DNA replication stop 5- Decatenation by topoisomerase

  27. The fidelity of DNA replication Control at two different stages: 1- Presynthetic control: Control at the incoming base 2- Proofreading:Substitution Determined by 3’ to 5’ exonuclease activities

  28. Replication in Eukaryotes

  29. Replication in EukaryotesThe differences between Pro and Eu The Replication rate in eukaryotes could be low at least because: 1- DNA is so long 2- There are many physical barriers BUT How to overcome this problem? Having hundreds of origins

  30. ARS (Autonomous replicating sequence) There are hundreds of Origin in eukaryotic genome, each is called ARS

  31. Licensing factor There is a 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

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

  33. Different Stages • Recognition of the ARS sequence by ORC • Initiation by DNAP α • Elongation by replisome • Termination by telomerase

  34. Elongation Nucleosome problem

  35. End Replication Problem

  36. Toxins and antibiotics that inhibit replication • Mitomycine: makes cross link between the two strands of DNA, preventing them of being template • Nalidixic acid: Prevents gyrase activity

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