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CMB Lehrer Replication Lecture 2018-1

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CMB Lehrer Replication Lecture 2018-1 CMB 621 – Fall 2018 DNA Replication – Part 1 Repair and Recombination Axel Lehrer Assistant Professor Tropical Medicine, Medical Microbiology and Pharmacology John A Burns School of Medicine, UH Manoa Before we tackle DNA replication… How do we even know it is the heritable material passed through generations? HISTORY 1928 - Frederick Griffith Streptococcus pneumoniae HISTORY 1944 - Avery, MacLeod and McCarty HISTORY 1952- Hershey and Chase Why is DNA replication important to study and understand? In vivo Importance S Essential for vertical propagation of information S May fix mutations S May create mutations -promote fitness & diversity -may result in cell death -may be neutral Also utilized in horizontal DNA transfer Utilized in some viral replication methods as well… Rolling Circle Replication Figure 15.7 Copyright © 2010 Academic Press Inc. Watson and Crick 1958 - Meselson and Stahl Semi-Conservative Replication 0 1 2 3 Figure 6-4 Essential Cell Biology (© Garland Science 2010) Where is the beginning site of DNA replication? G S 1 (DNA synthesis) G2 Cytokinesis MITOTICMitosis (M) PHASE Origin of Replication -Dictated by a specific-sequence motif Also influenced by chromatin conformation E. coli Origin of Replication •Note the AT-rich sequence (69%+) •Note the recognition binding sites for initiator proteins •Above is but one such motif discovered… 14 Copyright © 2010 Academic Press Inc. Initial Denaturation E. coli Ori Recap • Multiple binding sites at OriC • Recruitment of DnaA creates torsional strain at adjacent AT-rich motifs • Denaturation allows for DnaC (helicase loader/inhibitor) to deliver DnaB (helicase) • Helicase expands the replication bubble and DnaG (primase) allows for fork establishment Figure 5-27 Molecular Biology of the Cell (© Garland Science 2008) Is the ori fixed? What would happen if the ori picked up a mutation? Side Note Plasmid Oris S The particular ori found in a plasmid dictates the copy-number S Early generation plasmids contained an ori that gave low copy- numbers per cell S Contemporary plasmids contain a high copy-number ori that maintains plasmids at 25-50 per cell… why not go higher? Prokaryote E. coli has 1 ori or Eukaryote Humans have or approximately both? 30,000 - 50,000 Otherwise 30 days Figure 5-26 Molecular Biology of the Cell (© Garland Science 2008) Eukaryotic oris are found in clusters, ranging from 10-300 kb apart Different oris are utilized at different periods of the S phase Euchromatin oris are activated earlier than heterochromatin, as shown by examining replication of X chromosomes and comparing the timing of replication for housekeeping vs. less active genes Timing of Replication in Yeast Kinase activity at the S-phase leads to the degradation of initiator factors until the next round of the cell cycle While canonical human oris have been hard to elucidate, some appear very similar to the yeast ORC sequence Figure 5-36 Molecular Biology of the Cell (© Garland Science 2008) Ori is denatured to reveal a replication bubble, which then allows 2 forks to become established… Prokaryote Eukaryote Ori, Initiator Proteins, Bubble, Forks… What drives separation of the fork? Helicase = Mcm2-7 ATP is utilized Denatures ~ 1,000 bp/sec Composed of 6 identical subunits (in bacteria) These units have 3 different conformations Figure 5-14 Molecular Biology of the Cell (© Garland Science 2008) https://youtu.be/d_9VBgrDLUg We know it proceeds in a bi-directional fashion… But, intact dsDNA in front of fork builds torsional strain… Figure 5-25 Molecular Biology of the Cell (© Garland Science 2008) Type I DNA topoisomerases Reversible nucleases that transiently attach themselves to one strand of DNA Thereby creating a nick Torsional strain naturally resolves itself The energy of the phosopho- diester bond is retained in the transient complex Therefore no energy is needed and the rxn is rapid Figure 5-22 Molecular Biology of the Cell (© Garland Science 2008) Side Note Topo I TA Quick Cloning SSBP - Stabilizing Proteins RPA = replication protein A Figure 5-16 Molecular Biology of the Cell (© Garland Science 2008) SSBP helps to minimize inhibitory hairpin structures and mutations, and exposes unpaired bases Figure 5-17 Molecular Biology of the Cell (© Garland Science 2008) Now the DNA template strand is available for complementary synthesis… How does DNA pol know where to start synthesis? The leading strand only needs 1 primer for synthesis The lagging strand requires ribonucleotide primers at intervals of 100-200 nucleotides (eukaryotes) Notice that it reads the template 3’- 5’… but it synthesizes the nascent strand 5’- 3’ Why is a RNA primer used for DNA replication? Figure 5-11 Molecular Biology of the Cell (© Garland Science 2008) Direction of Synthesis Besides providing evidence for RNA-based early life de novo (new) synthesis can be error- prone, therefore it is better to come back later, remove the primer, and insert correct DNA bases Primers are marked as “suspect” If the cell used DNA primers, there is a greater chance of permanent incorporation of the errors By using RNA primers, these mutational hotspots will be subsequently removed DnaG – DNA Primase S Associates as a trimer with DnaB (helicase) S Tends to initiate synthesis at CTGs S 3 domains S Zinc BD S Helicase BD S RNA polymerase DNA Primase Regulation Redox in DNA primase regulates initiation (ox) and termination of priming (red) Model for primase product truncation, where primer-template handoff to the [4Fe4S] signaling partner, polymerase α in vivo, is regulated by DNA charge transport The Star DNA Polymerase S Many, many different types amongst various organisms S Its job is to produce complementary strands… with high-fidelity (usually) S But like many DNA scanning proteins, it has a propensity of falling off, so… Sliding Clamp = processivity - delivered by the clamp loader (Replication Factor C 1-5 in euks) - fixes DNA poly to the template, but releases it once the complex hits a dsDNA region in front of it Figure 5-18b Molecular Biology of the Cell (© Garland Science 2008) In eukaryotes the sliding clamp is called PCNA = homotrimer Proliferating Cell Nuclear Antigen aka – a processivity (1000x more) factor for DNA pol Figure 5-18c Molecular Biology of the Cell (© Garland Science 2008) https://youtu.be/5A77R3q0yZQ DNA (and RNA) is always synthesized in the 5’- 3 direction •Deoxy(ribo)nucleoside triphosphates are the building blocks •Hydrolysis of the phosphoanhydride bond releases part of the energy for the synthesis •The additional energy comes from the breakdown of the Note which phosphate resulting pyrophosphate group is incorporated? Figure 5-4 Molecular Biology of the Cell (© Garland Science 2008) Energetically, 3’ to 5’ synthesis will not suffice Figure 5-10 Molecular Biology of the Cell (© Garland Science 2008) Could you explain the components and process? Minimal Rates: Prokaryotic synthesis proceeds at 500- 1000 bases per second Eukaryotic synthesis proceeds at ~50 bases per second in vitro Taq synthesizes at 10- 45 bases per second One strand (leading) is made continuously and the other (lagging) is made discontinuously… Therefore replication is considered semi-discontinuous Prokaryotic Okazaki = 1 - 2 kb Eukaryotic Okazaki = 0.1 - 0.2 kb Notice that a bubble consists of forks that are inverted mirror images of each other Figure 6-12 Essential Cell Biology (© Garland Science 2010) The Replication Fork Is Asymmetrical At the replication fork the two newly synthesized strands are of opposite polarity…this clearly leads to logistical problems here since synthesis only proceeds in one direction No problem here though Notice the problem of the divergent polymerase movement? The replisome actually does stay intact… how? Sliding Trombone Model Figure 5-19a Molecular Biology of the Cell (© Garland Science 2008) https://youtu.be/-mtLXpgjHL0 https://youtu.be/4jtmOZaIvS0 Questions? Can we map it all out? Where are we? Primer Removal E. coli model DNA polymerase doesn’t start DNA synthesis de novo. The primer is RNA (about ~11 nucleotides in eukaryotes or ~5 nucleotides in prokaryotes) The primer is made by Primase, an RNA polymerase Pol III falls off and replaced The primer then has to be removed: by Pol I Pol I has 5- 3 exonuclease activity with which it cuts out the primer – as it Pol I does that it fills in the gap with DNA removes RNA primer In eukaryotes, FEN1 removes the and replaces it with DNA primer and new DNA is laid down by Pol d (it created a flap for FEN1) NOTICE DNA ligase then repairs the gap THIS!!!! Prokaryote DNA Pols S Pol I S Last pol, it removes previous Okazaki primer S 20 bases/sec, synthesizes the first ~ 400 S Involved in DNA repair as well S Pol III S Major pol for synthesis, ~1,000 bases/sec S Pol II S Involved in repair, a back up for pol III Eukaryotic Pols S a (+ primase) • Primase synthesizes ~10 RNA bases, then pol synthesizes the first ~15 DNA bases • Primarily initiates lagging strand synthesis • No exonuclease activity, but ~30,000/cell • e - Performs leading, (maybe more regulatory than CatalytiC?) • d (+ PCNA) • Greater processivity than above •Lagging strand extension, must be Constantly reloaded •Has 3’-5’ exonuclease activity Sg - mitoChondrial DNA repliCation Examples of Eukaryotic DNA Pols Eukaryotic DNA Pols S Eukaryotic DNA Pols S We’ve mentioned processivity, which means? We also need to address fidelity, which is? How does fidelity relate to 3’- 5’ exonuclease activity? Limiting Mutations Correct incoming base is a better fit Before covalent bond formation DNA pol undergoes a conformational change that can destabilize incorrect base pairing 3’- 5’ exonuclease activity Figure 5-8 Molecular Biology of the Cell (© Garland Science 2008) DNA Polymerase is Self-Correcting There are going to be mistakes, (mutations if they are not corrected) Mistakes are corrected by the 3’- 5 proofreading exonuclease activity of the polymerase (pol III, e and d) Initially, the mutation rate approaches 1 per 107 nucleotide pairs But the actual mutation rate approaches 1 per 109 nucleotide pairs -- other repair mechanisms (DNA mismatch repair) keep the mutation rate down.
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