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UIUC MCB 450 - Lecture 22 MCB450-F15 RF

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PowerPoint PresentationSlide 2Slide 3Replication proceeds 5’ to 3’The substrates for DNA replication are dNTPsSlide 6Slide 7DNA Pol I Core (Klenow Fragment)Slide 9Slide 10Slide 11The 2’-OH of ribonucleotides does not fit in the active site.Slide 13Slide 14Importance of Being ProcessiveSlide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32The Eukaryotic Cell CycleSlide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41•DNA replication Overview•Replication in Prokaryotes•Replication in Eukaryotes•Telomerase1Lecture 22 - Chapter 34DNA ReplicationDNA replication: Strand separation  copying of each strand.21. Four deoxynucleoside triphosphates and Mg2+ are required. (dATP, dCTP, dGTP and dTTP)2. A template strand is used to direct DNA synthesis.3. A primer from which the new strand grows must be present.4. Many DNA polymerases have nuclease activity that allows for the removal of mismatched bases.Key Characteristics of DNA Synthesis Are:___3Replication proceeds 5’ to 3’New strands is synthesized from 5’ to 3’ but template DNA is read from 3’ to 5’(DNA in the lab/RNA in vivo)4The substrates for DNA replication are dNTPsHow many high-energy bonds?5Strand Elongation ReactiondNTP(dATP, dCGP, dGTP, dTTP)6DNA polymerase in E. coli7DNA Pol I Core (Klenow Fragment)83 Activities:•Polymerase•3’ to 5’ exonuclease•5’ to 3’ exonuclease9Two divalent cations participate in the Polymerization Reaction10Shape selectivity of DNA polymerase11The 2’-OH of ribonucleotides does not fit in the active site.12Proofreading Activity of DNA Polymerase I13DNA Polymerase III has the highest processivityProperties of DNA polymerases in E. coli14Importance of Being Processive•It takes about 1 msec for a DNA polymerase to add a base (~ 1000 additions/sec).•It takes about 1 min for a polymerase to release and rebind DNA•So to copy a 5000 base DNA it takes a highly processive enzyme (holo-Pol III) only a few seconds, whereas several hours would be needed for a poorly processive enzyme to copy this DNA. 15Steps in replication in Prokaryotes- Unwinding of DNA Helix (DNA gyrase)- Strand separation (helicases/DnaB)- Binding of single-strand DNA-binding proteins (SSB) - Primer synthesis (primase)- DNA synthesis 5’ to 3’ (reads template 3’ to 5’) (pol III and I)- DNA fragment ligation (ligase)- Supercoiling16Topoisomerase II (Gyrase)17The origin of replication (oriC locus) in E. coli.18Pre-priming Complex(Helicase)19DNA Helicase (DnaB)Lagging strand20Primase (DnaG)RNA primer21Priming(Primosome)22Replication is BidirectionalBidirectional ReplicationoriCReplication eye/bubble23Replication is semi-discontinuous24gyraseHelicaseprimaseReplication fork25The DNA polymerase III holoenzyme26The Trombone Model27Actions of DNA Polymerase III and Primase28Actions of DNA polymerase I29DNA Pol I Removes RNA primer (5’ to 3’ exonuclease activity) and fills the gap between Okasaki fragmentsDNA Ligase seals the gap between Okasaki fragmentsDNA Ligase Reaction3’5’5’3’Okazaki-1Okazaki-2Phosphodiester bond link the two Okazaki fragmetns to each other3031Proteins involved in DNA Replication in E. coli(helicase)contrahelicasecontrahelicase32The Eukaryotic Cell Cycle33Eukaryotic cells contain a number of different DNA Polymerases34Multiple-Replicon Model of Eukaryotic Chromosomal DNA35The End Replication ProblemFirst generation3637What are Telomeres?TTAGGG-repeats38How are ends of chromosomes replicated (primer gaps filled)?Telomerase(reverse transcriptase)39Telomerase extends the 3’


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UIUC MCB 450 - Lecture 22 MCB450-F15 RF

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