Lecture 22 Chapter 34 DNA Replica6on Heredity I am the family face Flesh perishes I live on Projec ng trait and trace Through me to mes anon And leaping from place to place over oblivion Thomas Hardy Julie Newdoll s pain6ng Dawn of the Double Helix composes the DNA duplex as human gures 1 Outline DNA replica6on Overview Replica6on in Prokaryotes Replica6on in Eukaryotes Telomerase 2 DNA replica6on Strand separa6on followed by the copying of each strand 3 5 Parental old strand 3 Replica6on fork Template D NA Template DNA 5 New strand New strand Parental old 5 3 5 3 3 Key Characteris6cs 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 ac6vity 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 Elonga6on Reac6on dNTP dATP dCGP dGTP dTTP 7 Strand Elonga6on Reac6on 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 ca6ons par6cipate in the Polymeriza6on Reac6on 11 Shape selec6vity of DNA polymerase 12 The 2 OH of ribonucleotides doesn t fit in the active site 13 Proofreading Ac6vity of DNA Polymerase I 14 Proper6es 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 replica6on in Prokaryotes Unwinding of DNA Helix DNA gyrase Strand separa6on 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 liga6on ligase Supercoiling 17 Topoisomerases prepare the double helix for unwinding Type II gyrase Text book error p 602 above gure 34 8 Figure 34 8 Therefore nega6ve posi6ve supercoils 18 The origin of replica6on 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 Replica6on is Bidirec6onal Bidirec6onal Replica6on oriC Replica6on eye bubble 24 Replica6on is semi discon6nuous 25 Replica1on fork primase Helicase gyrase 26 DNA polymerase moving Right Replica1on fork primase Helicase gyrase 27 The DNA polymerase III holoenzyme 28 The Trombone Model holoenzyme 29 Ac6ons of DNA Polymerase III and Primase Direc6on of replica6on 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 Ac6ons of DNA Polymerase III and Primase Direc6on of replica6on fork Parental Leading strand 3 5 RNA primer 3 OriC DNA Polymerase 5 5 3 5 3 3 5 3 Okazaki fragments RNA primer 5 Parental Lagging strand 31 Ac6ons of DNA polymerase I Direc6on of replica6on 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 ac6vity and lls 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 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 5 template Okazaki 1 Okazaki 2 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 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 5 3 template 33 DNA Ligase seals the gap between Okasaki fragments Okazaki 1 Okazaki 2 T C G C A T T C A T G G T A G C C T T A G 3 5 T A G C T G G T A 5 3 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 5 template 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 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 5 3 template 34 DNA Ligase Reac6on 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 Replica6on of Prokaryo6c DNA 37 The Terminator Region Contains One Way Ter Sites Ter 38 Proteins involved in DNA Replica6on in E coli helicase contrahelicase 39 Di erences in Replica6on Mechanisms Prokaryotes DNA Eukaryotes DNA 4 6 million bp 1 chromosomes circular 1 origin Occurs through cell division cycle Rate of 1000 nucl sec Okazaki fragments of 1000 bp 6 billion bp 23 chromosome pairs linear 30 000 origin Limited to S phase Rate of 50 nucl sec Okazaki fragments of 100 200 bp 40 The Eukaryo6c Cell Cycle 41 Eukaryo6c cells contain a number of di erent DNA Polymerases 42 Need not know the details just appreciate the major points mentioned Mul6ple Replicon Model of Eukaryo6c Chromosomal DNA 43 Assembly of pre ini6a6on complex ORC is required to ini6ate DNA replica6on G1 phase 1 Preini6a6on complex assembled during G1 Replica6on licensing factors At end of G1 phase 2 Replica6on licensing factors destroyed un6l next G1 phase S phase 3 Polymerases and auxilliary factors assemble on preini6a6on complex during S phase 44 The End Replication Problem First genera6on 45 …
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