Lecture 22 Chapter 34 DNA Replication Heredity I am the family face Flesh perishes I live on Projecting trait and trace Through time to times anon And leaping from place to place over oblivion Thomas Hardy Julie Newdoll s painting Dawn of the Double Helix composes the DNA duplex as human figures 1 Outline DNA replication Overview Replication in Prokaryotes Replication in Eukaryotes Telomerase 2 DNA replication Strand separation followed by the copying of each strand 3 5 Parental old strand 3 Replication fork Template D NA Template DNA 5 New strand New strand Parental old 5 3 5 3 3 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 4 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 5 The substrates for DNA replication are dNTPs How many high energy bonds 6 Strand Elongation Reaction dNTP dATP dCGP dGTP dTTP 7 Strand Elongation Reaction 3 3 to 5 phosphodiester linkages 5 3 8 DNA polymerase in E coli 9 DNA Pol I Core Klenow Fragment Fingers Thumb Incoming Nucleotide Template Bend in Template Template Base Error rate of DNA polymerase 10 4 to 10 5 Primer 10 Two divalent cations participate in the Polymerization Reaction 11 Shape selectivity of DNA polymerase 12 The 2 OH of ribonucleotides doesn t fit in the active site 13 Proofreading Activity of DNA Polymerase I 14 Properties of DNA polymerases in E coli Kcat Processivity 3 200 500 000 DNA Polymerase III has the highest processivity 15 Importance of Being Processive It takes about 1 msec for a DNA polymerase to add a base so 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 PolIII only a few seconds whereas several hours would be needed for a poorly processive enzyme to copy this DNA 16 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 17 Topoisomerases prepare the double helix for unwinding Type II gyrase 18 The origin of replication oriC locus in E coli 19 Prepriming Complex Helicase 20 5 3 DNA Helicase DnaB 5 Lagging strand 21 Primase DnaG RNA primer 22 Priming Primosome 23 Replication is Bidirectional Bidirectional Replication oriC Replication eye bubble 24 Replication is semi discontinuous 25 Replication fork primase Helicase gyrase 26 DNA polymerase moving Right Replication fork primase Helicase gyrase 27 The DNA polymerase III holoenzyme 28 The Trombone Model holoenzyme 29 Actions of DNA Polymerase III and Primase Direction of replication fork Leading strand 3 5 RNA primer 3 5 OriC 3 5 3 3 5 5 3 Okazaki fragments RNA primer 5 Lagging strand 30 Actions of DNA Polymerase III and Primase Direction of replication fork Parental Leading strand 3 5 RNA primer 3 5 OriC DNA Polymerase 3 5 3 3 5 5 3 Okazaki fragments RNA primer 5 Parental Lagging strand 31 Actions of DNA polymerase I Direction of replication fork Leading strand 3 5 5 3 OriC gap 5 3 3 5 3 5 Okazaki fragments Lagging strand 32 DNA Pol I Removes RNA primer 5 to 3 exonuclease activity and fills the gap between Okasaki fragments Okazaki 1 RNA primer 1 Okazaki 2 5 T A G C T G G T A U C G C A U U C A U G G T A G C C T T A G 3 5 3 5 3 A T C G A C C A T A G C G T A A G T A C C A T C G G A A T C 5 3 template Okazaki 1 Okazaki 2 3 5 T A G C T G G T A U C G C A U U C A T G G T A G C C T T A G 3 5 5 3 A T C G A C C A T A G C G T A A G T A C C A T C G G A A T C 5 3 template 33 DNA Ligase seals the gap between Okasaki fragments Okazaki 1 Okazaki 2 5 T A G C T G G T A T C G C A T T C A T G G T A G C C T T A G 3 5 3 3 5 3 A T C G A C C A T A G C G T A A G T A C C A T C G G A A T C 5 template 3 5 T A G C T G G T A T C G C A T T C A T G G T A G C C T T A G 35 5 3 A T C G A C C A T A G C G T A A G T A C C A T C G G A A T C 5 3 template 34 DNA Ligase Reaction Okazaki 2 3 Okazaki 1 5 5 3 Phosphodiester bond link the two Okazaki fragmetns to each other 35 DNA replication 1 Replisome 2xPol III Synthesizes both Leading and Lagging Strands at Replication Fork Pol III Pol III Okazaki Fragments 2 After Passage Pol I Removes RNA primer and Completes DNA Synthesis 3 DNA Ligase Seals Nicks Nick 3 5 P OH 5 3 5 3 5 3 36 Replication of Prokaryotic DNA 37 The Terminator Region Contains One Way Ter Sites Ter 38 Proteins involved in DNA Replication in E coli helicase contrahelicase 39 Differences in Replication Mechanisms Prokaryotes DNA Eukaryotes DNA 4 6 million bp 1 chromosomes circular 1 origin Occurs through cell division cycle 6 billion bp 23 chromosome pairs linear 30 000 origin Limited to S phase Rate of 1000 nucl sec Rate of 50 nucl sec Okazaki fragments of 1000 Okazaki fragments of 100 bp 200 bp 40 The Eukaryotic Cell Cycle 41 Eukaryotic cells contain a number of different DNA Polymerases 42 Need not know the details just appreciate the major points mentioned Multiple Replicon Model of Eukaryotic Chromosomal DNA 43 Assembly of pre initiation complex ORC is required to initiate DNA replication G1 phase 1 Preinitiation complex assembled during G1 Replication licensing factors At end of G1 phase 2 Replication licensing factors destroyed until next G1 phase S phase 3 Polymerases and auxilliary factors assemble on preinitiation complex during S phase 44 The End Replication Problem First generation 45 46 What are Telomeres TTAGGG repeats 47 How are ends of chromosomes replicated primer gaps filled Telomerase reverse transcriptase reverse …
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