Lecture 22 Chapter 34 DNA Replication DNA replication Overview Replication in Prokaryotes Replication in Eukaryotes Telomerase 1 DNA replication Strand separation copying of each strand 2 Key Characteristics of DNA Synthesis Are 1 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 3 Replication 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 4 The substrates for DNA replication are dNTPs How many high energy bonds 5 Strand Elongation Reaction dNTP dATP dCGP dGTP dTTP 6 DNA polymerase in E coli 7 DNA Pol I Core Klenow Fragment 3 Activities Polymerase 3 to 5 exonuclease 5 to 3 exonuclease 8 9 Two divalent cations participate in the Polymerization Reaction 10 Shape selectivity of DNA polymerase 11 The 2 OH of ribonucleotides does not fit in the active site 12 Proofreading Activity of DNA Polymerase I 13 Properties of DNA polymerases in E coli DNA Polymerase III has the highest processivity 14 Importance 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 15 Steps 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 Supercoiling 16 Topoisomerase II Gyrase 17 The origin of replication oriC locus in E coli 18 Pre priming Complex Helicase 19 DNA Helicase DnaB Lagging strand 20 Primase DnaG RNA primer 21 Priming Primosome 22 Replication is Bidirectional Bidirectional Replication oriC Replication eye bubble 23 Replication is semi discontinuous 24 Replication fork primase Helicase gyrase 25 The DNA polymerase III holoenzyme 26 The Trombone Model 27 Actions of DNA Polymerase III and Primase 28 Actions of DNA polymerase I DNA Pol I Removes RNA primer 5 to 3 exonuclease activity and fills the gap between Okasaki fragments DNA Ligase seals the gap between Okasaki fragments 29 DNA Ligase Reaction Okazaki 2 3 Okazaki 1 5 5 3 Phosphodiester bond link the two Okazaki fragmetns to each other 30 31 Proteins involved in DNA Replication in E coli helicase contrahelicase 32 The Eukaryotic Cell Cycle 33 Eukaryotic cells contain a number of different DNA Polymerases 34 Multiple Replicon Model of Eukaryotic Chromosomal DNA 35 The End Replication Problem First generation 36 37 What are Telomeres TTAGGG repeats 38 How are ends of chromosomes replicated primer gaps filled Telomerase reverse transcriptase 39 Telomerase extends the 3 strands 40 41
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