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SINTESIS DNA

REPLIKASI DNA. SINTESIS DNA. Study Objective. 1 . Understand how the following terms apply to DNA replication: template , complementary base pairing, origin, bi-directional, theta structure, replication fork, semi-conservative.

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SINTESIS DNA

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  1. REPLIKASI DNA SINTESIS DNA

  2. Study Objective • 1. Understand how the following terms apply to DNA replication: template, complementary base pairing, origin, bi-directional, theta structure, replication fork, semi-conservative. • 2. Know how the following enzymes function in leading and lagging strand replication: helicase, ssDNA binding protein, primase, DNA polymerase III, DNA polymerase I. What is an Okazaki fragment? • 3. What is proofreading? • 4. Understand the problem of replicating the ends of linear DNA. Understandhow telomerase solves that problem for eukaryotic chromosomes.

  3. Flow of information replication DNA DNA transcription  RNA translation  protein

  4. cell cycle

  5. Overview of bacterial DNA replication single origin (in bacteria) bidirectional theta structures replication fork semi-conservative TB

  6. Bidirectional

  7. bacterial DNA replication TB bidirectional origin (start point) bacterial chromosome

  8. theta structure two replication forks TB

  9. konservatif semikonservatif dispersif

  10. semi-conservative * * * + * TB

  11. DNA Replication Let’s meetthe team… • Large team of enzymes coordinates replication

  12. KEPERLUAN REPLIKASI DNA • TEMPLATE (CETAKAN) • PRIMER : 3’-OH - PERPANJANGAN • PREKURSOR : dNTP • Enzim

  13. Replication I’d love to behelicase & unzipyour genes… • Unwind DNA • helicase enzyme • unwinds part of DNA helix • stabilized by single-stranded binding proteins helicase single-stranded binding proteins replication fork

  14. Key Enzymes helicase ssDNA binding protein primase DNA polymerase III DNA polymerase I DNA ligase TB

  15. Helicase Unwinds duplex DNA 3' 5' TB

  16. ssDNA binding protein binds to and stabilizes ssDNA ssDNA binding protein 3' 5' prevents base pairing TB

  17. Template • A sequence of DNA or RNA that directs the synthesis of a complementary sequence 3' 5'

  18. Important facts All DNA polymerases require a primer DNA is synthesized 5' to 3' TB

  19. Primer • The initial segment of a polymer that is to be extended on which elongation depends

  20. primase synthesizes a short RNA primer using a DNA template primase 3' 5' RNA primer (a short starting sequencemade of RNA)

  21. Polimerase • Memerlukan primer dancetakan DNA • Polimerisasidiperpanjangpada 3’ • Aktivitaseksonuklease 3’ -5’, berfungsisebagaiproofreading (pembetulan pembacaan) • Aktivitaseksonuklease 5’-3’ untukmenghilangkan primer

  22. DNA polymerase III Synthesizes DNA from a DNA template and proofreads TB

  23. REAKSI PERPANJANGAN

  24. DNA polymerase I Synthesizes DNA from a DNA template and removes RNA primers. TB

  25. DNA ligase MenyambungduafragmenOkasaki denganmembentukikatanfosfodiester antara3’-OH fragmen 1 dengan 5’-P fragmen 2 DNA ligase TB

  26. Replicationfork template strands

  27. DNA polymerase III 3 3 3 3 3 3 3 3 3 3 3 growing replication fork growing replication fork 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Replication fork / Replication bubble leading strand lagging strand leading strand lagging strand leading strand lagging strand

  28. Leading strandsynthesis 3' ssDNA binding proteins RNA primer helicase 5' TB

  29. Leading strandsynthesis 3' ssDNA binding proteins DNA Pol III helicase 5' TB

  30. Lagging strand synthesis (discontinuous) 3' helicase Okazaki fragment (~1000 bases) (primase) ssDNA binding proteins Pol III 5' TB

  31. Primer removal pol III 3' 5' pol I 5’ to 3’ exonuclease activity pol I TB

  32. Ligase DNA • MenyambungduafragmenOkasakidenganmembentukikatanfosfodiesterantara 3’-OH fragmen 1 dengan 5’-P fragmen 2

  33. Ligation DNA ligase TB

  34. 5 3 5

  35. Lokus awal replikasi (Ori)

  36. INISIASI

  37. Prokaryotic vs. Eukaryotic • Bacterial cells have one giant looped chromosome • Replication can occur in one or two directions • Oneorigin of replication • In Eukaryotes manyorigins of replication exist • These form replication bubbles • Eventually bubbles meet and replication is done • Replication forks - where DNA is opened up

  38. REPLIKON E.coli

  39. REPLIKON MAMALIA

  40. 1 2 3 4 What does it really look like?

  41. TELOMER

  42. 3 3 3 3 5 5 5 5 Houston, we have a problem! Chromosome erosion DNA polymerase I growing replication fork DNA polymerase III RNA Loss of bases at 5 endsin every replication • chromosomes get shorter with each replication • limit to number of cell divisions?

  43. Replication of the ends of linear DNA newly synthesized DNA RNA primer 3' 5' template Since all known DNA polymerases need a primer, how are the ends of linear DNA replicated in eukaryotes? TB

  44. TELOMER Telomerterletakdiujungkromosom. Umumnyaberkode TTAGGG/AATCCC. Mengawasiprosespembelahanselsupayatidakadarangkaian DNA yang hilang. Telomermencegahkromosomtidaksalingmenempeldanterbelit yang bisamenyebabkanurutan gen kacaudanterjadimalfungsiselygberakibatkarsinogenikataumati. Sebelumselmembelah, kromosomlebihdulumenggandakandirinya ( REPLIKASI), selanjutnyaberpisah. Padareplikasi, enzim DNA polimerasemengcopydua single strand DNA denganbantuan PRIMER, suatu RNA. SaatlahirpanjangTelomer 8.000 pasangbasa, saatusialanjuttinggal 3.000 bp. Setiap x seltubuhmembelah, 30 s.d 200bp hilangdrtelomer. Semakinpendektelomer, selsemakintua

  45. 3 3 3 3 5 5 5 5 Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap • limit to ~50 cell divisions growing replication fork telomerase Telomerase • enzyme extends telomeres • can add DNA bases at 5 end • different level of activity in different cells • high in stem cells & cancers -- Why? TTAAGGG TTAAGGG TTAAGGG

  46. Example GGGGTT GGGGTT GGGGTT (GGGGTT)n n = 20 - 200 Telomeres repetitive DNA at the end of linear eukaryotic chromosomes 5' TB

  47. AACCCCAAC telomerase Telomerases are enzymes that add DNA repeats to the 3' end of DNA. Telomerases are composed of protein and an RNA molecule that functions as the template for telomere synthesis.

  48. Human telomerase • Telomerase = ribonucleoprotein complex • Ribo = ribosomal/RNA association • Nucleo = nuclear localization • Protein = contains a protein • Responsible for maintaining telomere length in eukaryotic chromosomes • Main components: • Telomerase reverse transcriptase • Human telomerase RNA (hTR)

  49. Human telomerase (2) • Telomeres – repeated regions at the end of eukaryotic chromosomes • hTR is the template for the repeated region

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