Lecture 14 - DNA replication in bacteria 1 Last Time Virus...

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Unformatted text preview: DNA replication in bacteria 1 Last Time Virus genomes Composition and structural variety! Bacteria genomes Organization of DNA (landmarks and features) Higher order structures Eukaryotic genomes Organization of DNA (landmarks and features) Higher order structures 2 DNA replication in bacteria The semi-conservative model of DNA replication Bacterial replication mechanics Initiation Elongation Termination An in-depth view of Polymerases 3 How is DNA replicated? "I t h as n o t e s c ap e d o u r n o t ic e t h at t h e s p e c ific p a i r i n g we h ave p o s t u l ate d i mme di ate l y s ug ge s t s a p o s s i ble c o py i n g me c h a n i sm f o r t h e g e ne t ic m ate r i a l ." - J.D. Watson and F.H.C. Crick Nature (1953)171: 738 http://www.sciencemuseum.org.uk 4 Three hypothetical models of DNA replication Semi-conservative Conservative Dispersive 5 1958 Classic experiment to distinguish between 3 replication models Franklin Stahl Matthew Meselson “t h e m o st b e a u ti f u l exp e r i m e nt i n bi o l o g y ” 6 Conservative HH Expected results HH shift HH LL LL HH second generation Semiconservative Expected results shift HH HH HL HL LH LH LL LL HL LL H L Dispersive HH Expected results HH shift mixed mixed HL all mixed HL Figure 20.3 7 The DATA: first generation Semiconservative Expected results second generation shift HH HH HL HL LH LH LL LL HL LL H L Generation after 14N addition Light H/L Heavy 4th 3rd 2nd 1st 8 DNA replication is semi-conservative 1st generation 2nd generation 9 DNA replication in bacteria Initiation Elongation Termination 10 Origin of Replication (oriC in E. coli) 11 DNA motifs in the oriC region 12 oriC portion of chromosome DnaA protein, et al binds and... contorts DNA helicase binds ‘unzips’ DNA 13 DNA gyrase (topoisomerase) Single Stranded Binding Proteins (SSBPs) Helicase 14 DNA gyrase introduces negative coils to relax overwound region 15 DNA polymerase III primer Primase Direction of replication 16 Adding nt to the 3’OH of growing strand dATP 17 DNA polymerase (DNAP) Synthesis moves in 5’-3’ direction DNAP can only add to existing nt (cannot start synthesis from scratch) 18 If DNA pol can only move in a 5’-3’ direction, how does DNA pol synthesize DNA on the other template strand? 19 Lagging strand synthesis is discontinuous Okazaki fragments 20 But our DNA doesn’t have little pieces of RNA stuck in it, right? 21 RIGHT! Another type of DNAP (DNAP I in E. Coli) removes and replaces primers with DNA But….how are Okazaki fragments stitched together? 22 DNA ligase seals the nicks in the backbone between two Okazaki fragments on the lagging strand Okazaki fragments DNA ligase joins the 5’ phosphate of one DNA molecule to the 3’ OH of another to seal fragments together 23 initiation Terminating DNA synthesis Ter regions TUS proteins bind Ter region, stops helicase Fig. 21.27 24 Decatenation of Daughter DNAs topoisomerase 25 What’s wrong with this picture? Helicase DNA pol III leading strand Primase lagging strand DNA pol I Ligase 26 DNA polymerases in E. coli 5 DNA polymerases: DNAP III*, DNAP I - involved in replication DNAP II, IV, V - repair 27 Core Clamp loader DNAP III has 10 subunit. “Core subunits” contain the polymerizing ! subunits. http://www.canadacool.com/COOLFACTS/NEW%20BRUNSWICK/NBPHOTOS/Lobster.jpg 28 " - Clamp . “Clamps” ! subunit of DNA polymerase III onto DNA 29 helicase DNA gyrase primase 30 Ungraded problems Look over Chapter 11 S3 Try Chapter 11 C2, C3, C7, C8, C10a*, C16 31 Summary Meselsohn and Stahl demonstrated that DNA is replicated semi-conservatively Initiation of bacterial DNA replication requires binding of proteins at the oriC Many proteins act together at the replication fork to drive DNA synthesis Tus proteins bound to Ter sequences, opposite of the oriC, halt replication DNAP III has multiple subunits, and polymerizes DNA on the leading and lagging strand at the same time 32 Next Time DNA replication in eukaryotes http://biology.kenyon.edu/courses/biol114/Chap01/chrom1.gif http://cmb.duke.edu/images/rotating/counterpic2.jpg 33 ...
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