ls3-dna-replication-lec-11-fill-in-2

ls3-dna-replication-lec-11-fill-in-2 - prokaryo8c...

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Unformatted text preview: prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! DNA replica.on •  Uncoiling helix •  Strand separa.on (breaking H‐bonds between complementary strands) •  Synthesis of new strands by complementary base pairing LS3‐2 week 6 discussion Page 1  Where does replica.on start?  Uncoiling the helix + strand separa.on HELICASE gene = __________________ Depends on ______ for energy TOPOISOMERASES SINGLE STRANDED BINDING PROTEINS (SSBPs) Func.ons (other than binding to single strands of DNA): •  •  •  •  •  nick and allow rota.on of DNA •  relieves strain fromstrand separa.on •  2 types: Type I: single strand breaks Type II: double strand breaks  Replica8on Fork(s) LEADING STRAND – con.nuous 5’3’ LAGGING STRAND – discon.nuous 5’3’ Wait!!! Before DNA polymerase can begin DNA synthesis, something else needs to happen PRIMING Why?? DNA polymerase III is only able to join the 5’‐carbon’s phosphate group to the 3’‐carbon’s hydroxyl (OH) group of a nucleo.de already in the chain; in other words, it can only catalyze NT addi)on to pre‐exis)ng ones  Priming DNA PRIMASE gene = ___________________ Type of nucleic acid ? Length __________ prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! prokaryo8c! DNA replica.on •  Uncoiling helix •  Strand separa.on (breaking H‐bonds between complementary strands) •  Synthesis of new strands by complementary base pairing LS3‐2 week 6 discussion Page 2  Synthesis of new DNA strands DNA POLYMERASE III gene= ___________ Core enzyme Holoenzyme β‐subunit of DNA POL III • DIMER forms a SLIDING CLAMP • =tether that keeps DNA pol III associated with DNA • keeps DNA synthesis PROCESSIVE (finishes what it starts) • needs CLAMP LOADER (aka _____________) to bind DNA 3 proteins: α,θ,ε Func.on(s): polymerase + exonuclease ac.vity Components: lots more proteins Notable member: β‐subunit   Connec.ng Okazaki fragments • Need to get rid of RNA (primers) • Replace with DNA • Ligate pieces together Two enzymes can excise RNA primers: • RNase H • DNA polymerase I DNA POLYMERASE I ‐ mul.‐func.onal • 5’3’ exonuclease ac8vity = removes RNA primers • DNA polymerase ac8vity = fills in gaps with dNTPs • 3’5’ exonuclease proofreading ac.vity DNA polymerase can only add new NTs to exis.ng ones So the last step requires another enzyme: DNA LIGASE • ligates the 5’‐phosphate to the 3’‐OH • Requires a cofactor: ____or ____ (for energy ) Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments! Lec 11experiments! Lec 11experiments! Meselson & Stahl Experiment Significance: proved that DNA replica.on is semi‐conserva.ve Key technique: Cesium chloride (CsCl) density gradient centrifuga.on Methods/Procedure: • Grow bacteria for many genera.ons in media containing “heavy” nitrogen (isotope 15N) Why? So that (almost) all bacteria have genome containing bases with heavy nitrogen • Shi` bacterial culture from (1) to growth media containing “light” 14N Why? So that any newly synthesized DNA will have light nitrogen incorporated • A`er shi`ing to “light” growth media, isolate DNA from bacteria at different .me points (i.e. 10 min, 20 min, 40 min) Why? • Centrifuge isolated DNA in a CsCl (cesium chloride) gradient to separate DNA by density • Shine UV light to visualize banding of DNA LS3‐2 week 6 discussion Page 3 Results L H At genera.on “0” why do you only see DNA containing 15N? “0 and 4.1 mixed” significance? Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments! Lec 11experiments! Lec 11experiments! RNA primers – Size determina8on experiment Significance: showed discrete size of RNA primers (10‐12NTs long) Key technique: radioac.ve labeling of RNA primer with 5’ capping enzyme (guanylyl transferase) LS3‐2 week 6 discussion Page 4 Methods/procedure: • Isolate Okazaki fragments from bacterial cells (isolate total DNA, separate strands, purify short fragments) • Mix Okazaki fragments + guanylyl transferase(5’capping enzyme) + 32P‐GTP(radioac.ve GTP) Why? Will radioac.vely label 5’ end of the primer • Treat capped Okazaki fragment with Dnase Why? Will degrade DNA and you’ll be le` with labeled 32P‐GTP + RNA primer • Gel electrophoresis and autoradiography Why? Size determina.on (gel) and visualiza.on Results What’s up with lanes e‐g? RNA primers are transient – to see them you need to inhibit the enzymes that degrade them Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments!Lec 11experiments! Lec 11experiments! Lec 11experiments! Temperature sensi8ve mutants Why, oh why (and when), did we talk about them? LS3‐2 week 6 discussion Page 5 DNA Helicase Assay Significance: shows helicase is responsible for separa.ng DNA strands (at the replica.on fork) Methods/Procedure: • Genera.ng a… ((take a deep breath)) “radioac.vely‐labeled synthe.c single stranded DNA that only has its center region complementary to a region of a phage single stranded circular DNA” Why? Making a fake replica.on fork • Mix synthe.c/phage hybrid + dnaB protein (helicase) + ATP (for energy) Why? To test enzyme ac.vity • Run product on a polyacrylamide gel Why? To see if there was any enzyma.c ac.vity Results Before Aher rxn rxn ...
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This note was uploaded on 04/19/2011 for the course LS 2 taught by Professor Pires during the Spring '08 term at UCLA.

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