Molecular Biology I
Molecular Biology I MBioS 503
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This 23 page Class Notes was uploaded by Carol McDermott on Thursday September 17, 2015. The Class Notes belongs to MBioS 503 at Washington State University taught by Staff in Fall. Since its upload, it has received 16 views. For similar materials see /class/205938/mbios-503-washington-state-university in Molecular Biosciences at Washington State University.
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Date Created: 09/17/15
Course website httpmolecuIarbioscienceswsueduacademicscoursesmbi05503htm Suggested Reading 1 Schaeffer PM Headlam MJ Dixon NE 2005 Proteinprotein interactions in the eubacterial replisome IUBMB Life 2005 Jan571512 2 Neylon C Kralicek AV Hill TM Dixon NE 2005 Replication termination in Escherichia coi structure and antihelicase activity of the TusTer complex Microbiol Mol Biol Rev 2005 Sep693501 26 Simultaneous Replication Occurs via Looping of the Lagging Strand is subunlt slldlng clamp l Singiesiiandmi binding protelris Lagging sirand template Not shown pol I Iigase DnaA binds at the OriC DnaBHelicase unwinds helix SSBs prevent closure DnaG the primase RNA polymerase synthesises short RNA primers Association of core polymerase with template JContinuous DNA synthesis on leading strand Okazaki fragments on lagging strand processed by Poll and joined by DNA ligase What if the wrong base is put in 7 Pol III has 339 to 539 exonuclease activity 7 Polymerase backs up and removes incorrect base 7 Usually recognized by distortion in the helix DNA damage can halt replication DNA damage can nan replication Replication lurk advances on norma DNA Leaamg strand In 39 A replication fork stalls when it arrives at damaged DNA 39 After the damage has been repaired the primosome is required to reinitiate replication A stalled replication ark can leslarl Replication apparatus is inactivated l l EJlllllll lll 39 Damage is repaired and primosome binds 7 e 39 EIIEIJJI e m 1 1 I y Heplicalion resumes m quot lt77 n m l I I Recombination repairs a stalled fork Single 51mm invades quotom mher daughter duplex I I 04 Crossover is rescived I Repiicalmn resumes 0 The twisting problem Unwinding puts tension on molecule and introduces supercoils Gyrase topoisomerase removes supercoils Unwinding during replication The twisting problem Motion 7 D repilcaimg York a BOXAG Without free OHng 1 D I 03 rotation US32 Q quot Q Rotation an the direciionommvmnmg unreplicated region is overwound 0004 Topoisomerases laggingrslrand emplale DNA polymerase on leading strand newly synthesized DNA chain 3 Topoisomerase Types Tyge 1 Produce single stranded breaks in one of the DNA backbones Allows one of the phosphodiester bonds to rotate freely around the other relieving tortional stress Tyge 2 Produces double stranded breaks in the DNA backbone at the same time This is necessary to untangle replicated chromosomes 0Type removes Type I One strand 0 n e 5 pe rco39 I at DNA with one strand passed through broken and resealed two r a U me supercoils 0Type removes two supercoils at a time Both strands passed through each other and resealed V both strands oken Relaxed circle Baa n ooh 5m 5011 by w Figure 43 re b Molecular Cell Biology Sixlli Edition Molecular Cell Biology Sixlli Edition 2008 w H Freeman and Company e 2008 w H Freeman and Company Wrapping up chromosome replication 0 Termination meeting of two replication forks and the completion of daughter chromosomes Chromosome 0 Region 180 from ori contains replication fork traps Wrapping up chromosome replication Mm Termination meeting of two on replication forks and the completion of daughter chromosomes TLS monomers I Region 180 from ari contains replication fork traps Organization of the replication initiation and termination sites on the E calichromosome Replication Termination of the Bacterial Chromosome 0 One set of Ter sites arrest DNA forks progressing in the clockwise direction a second set arrests forks in the counterclockwise direction TerB TerA Replisome of E coli and mechanism of replication fork arrest by a TusTer complex A Lagging strand SSE protein DnaB hollcasa L m39 39 Leading strand 9 I Tm Laqqmq strand loop pol39 m RNA primer holocnzymc The Tus protein binds to ter sites and stops DnaB from unwinding DNA which causes replication to terminate Tus acts asymmetrically DNA accessible DNA blocked Replication proceeds Replication terminates The End Problem 0 How do you replicate at the ends of chromosomes Usually only a problem for eukaryotes 0 linear chromosomes 0 Use specialized enzyme called telomerase Some bacteria have linear chromosomes Borrelia burgdorferi Causes Lyme disease 5 phosphate and 3 OH at each end of chromosome make hairpins Replication from ori in middle not bidirectional Resolvase recreates hairpin ends ZSbp inverted repeat Replication initiates at internal origins Telomere resolvase recognition site Telomere resolvase cleaves and rejoins hairpin ends f quoti I l l f i ii A Summary 0 Some of the DNA replication proteins DNA Pollll DNA Poll DNA Ligase Primase DnaG Helicase DnaB 53 0 Replication termination Replication fork traps opposite oriC Ter sites DNA Polymerases are useful in the lab Polymerase 339 gt539 539 gt339 539 gt339 Exo Exo 99 E coli Pdl Nick Tra nslation Klenqurajgment Primer extension polishing ends a portlon of 01 I Taqul PCR error prone Pfu Pdl PCR accurate T7 Sequenase Sequencing reactions E coli DNA pol poA Easily cleaved into two fragments by proteinase large fragment Klenow fragment contains polymerase and 3 5 exonuclease proofreading domain Used in vitro for synthesis reactions DNA sequencing KlenOW fragment 58 kD small fragment 35 kD N lm 7 C c exonuclease polymerase 539339 exonuclease 3 5 site proofreading catalytic removes RNA primer amp quot10 bp DNA in vitro DNA synthesis using the Klenow fragment DNA pol I Experimental Uses a Fillin reaction to label recessed ends of DNA b DNA sequencing 32p 5 339 lt 539 No other bacterial polymerase can initiate at a nick
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