MBB 331 7 - Lecture notes 7 PDF

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1 Lecture 7: Prokaryotic DNA Replication  Overview  E. Coli DNA polymerases  DNA pol III  DNA pol I  Initiation of Replication  DNA Replication: Consider this  Meselson and Stahl experiment determined that DNA replication is semiconservative  Replication occurs in the 5’ to 3’ direction however the helix is antiparallel  Replication errors occur 1 in 10^10 base pairs  How is replication of the two strands coordinated?  How is replication made so accurate? 

Polymerases synthesize DNA in the 5’ to 3’ direction and in order for DNA to replicate, the two strands of the double helix must unwind (melt)

 Temperature Sensitivity (TS) in bacterial (yeast) culture  A TS mutant is able to replicate at ONE region of a temperature range but NOT at another  To make a TS mutant: o Introduce mutagens in cell culture of bacteria o Let culture grow o Temperature shift to find mutants that do not grow = CONDITIONAL LETHALS  Makes it possible to compare + contrast mutant phenotypes by adjusting the temperature so important genes can be identified and understood  Using BACTERIAL genetics to identify proteins involved in replication  Inability to replicate DNA is fatal for cell hence mutations in replication must be isolated as conditional lethals  EX. Temperature sensitive mutants in DNA replication can only function at permissive temperature of incubation (37 degrees Celsius) and are lethal at a higher, non-permissive/restrictive temperature (40 degrees Celsius)  A collection of such ts mutants has been identified in E. Colu which form the set of dna genes  E. Coli DNA replication mutants  Fast/Quick Stop mutants (type I): replication immediately stops after shift to 40 degrees Celsius o Mutants must be defective in ONGOING replication machinery  Slow Stop mutants (type II): following 40 degrees Celsius shift, replication stalls after one complete round of replication o Mutants defective in events INITIATING replication

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 BACTERIAL DNA polymerases  Kornberg (1958) discovered first DNA polymerase (DNA pol I) o Assumed it replicated bacterial genome  DeLuca and Cairns isolated DNA pol I mutant (DNA pol AI) o No DNA pol I activity but cells still alive o Suggested another DNA polymerase that could replicate bacterial genome (DNA pol II or III)  Kornberg: dnaE mutants abolished genome replication and caused lethality; dnaE = DNA pol III alpha subunit 

Three DNA pols have been studied in E. Coli: DNA pol I, II, and III (names reflect order of discovery) 1. All catalyze template-directed 5’ to 3’ synthesis from dNTP precursors 2. All require free 3’-OH (primer) 3. All possess 3’ to 5’ exonuclease activity (proofreading)

 DNA pols synthesize DNA in a PROCESSIVE manner  DNA polymerases bind the primer: template junction o Upon binding, non-processive DNA polymerase adds single dNTP to 3’ end of primer and is released from new primer: template junction o Contrast, processive DNA polymerase adds many dNTPs each time it binds to template  Beta clamp confers processivity  DNA polymerase is TEMPLATE-DIRECTED  DNA polymerase requires: o Template DNA strand o dNTPs o free 3’-OH provided by short stretch of RNA (A) 

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align pried template for sequential addition of incoming nucleotides

DNA polymerases require a primer strand and a template strand (i.e. primed template) As dNTPs are added to 3’-OH of primer strand, strand grows in 5’ to 3’ direction Incoming dNTPs are complementary to template strand (B) Insertion and postinsertion sites in DNA polymerase properly

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During replication of cellular DNA, free 3’-OH is provided by synthesis of short RNA primer by primase Primase: specialized RNA polymerase dedicated to making short RNA primers (5-10 nts long) on a ssDNA template

 Structure of the replication fork and semi discontinuous replication  Double helix unwinds and separated two strands forming replication fork: o Both daughter strands are synthesized 5’ to 3’ (elongation)  With respect to direction of fork movement: o One DNA polymerase moves in 5’ to 3’ direction on one strand o Another DNA polymerase moves 3’ to 5’ on other strand  This requires two different forms of synthesis: o DNA synthesis occurs continuously on leading strand in the 5’ to 3’ direction as DNA duplex is unwound o DNA synthesis occurs discontinuously on lagging strand in reverse direction relative to fork movement  Lagging strand synthesis o Polymerase must wait for a stretch of ssDNA (parental) to be exposed before synthesizing a fragment of DNA o Fragments of DNA synthesized in this manner re later joined together by DNA ligase to generate a complete lagging strand * therefore, DNA synthesis is semi-discontinuous  Reiji Okazaki o Showed discontinuous replication on lagging strand can be followed by adding brief radioactive labels which incorporate into newly synthesized lagging strand fragments of 1000 to 2000 bases long in prokaryotes  RNA primers o Leading strand only requires one priming event by primase o Lagging strand requires series of primers each about 10 bases long to make each Okazaki fragment  3’ to 5’ PROOFREADING exonuclease  DNA polymerases possess a proofreading function that improves the replication-error rate 1. When incorrect nucleotide is incorporated = rate of synthesis stalls 2. Mismatched 3’ end becomes single stranded – repositioning offers increased affinity for exonuclease active site = mismatch is removed 3. Once mismatched DNA removed, primer: template junction is restored and polymerization resumes

4  Structure of DNA polymerase reveals basis for accuracy

 5’ to 3’ Exonuclease Activity  DNA pol I has second exonuclease that degrades    

DNA in 5’ to 3’ direction (direction of synthesis) DNA pol I performs variety of clean up functions during replication, recombination and repair All involve trimming of ssDNA ends and removal f RNA primers or DNA lesions Functions at the same time as DNA pol I because same direction DNA or RNA strand in duplex is degraded while polymerase adds nucleotides behind it (concerted) = NICK TRANSLATION o Nick is missing phosphodiester bond in one strand

 KLENOW fragment of DNA pol I lack 5’ to 3’ exonuclease activity (commonly used in labs)  Protein fragment produced when DNA pol I is cleaved  Without its 5’ to 3’ exonuclease domain, DNA pol I has no DNA or RNA degradation activity  Synthesis of dsDNA from ss templates  Fill in receded 3’ ends  Digest 3’ overhangs  Highly processive  Taq DNA pol is a Klenow Fragment  In VIVO roles of E. Coli DNA polymerases  Main replicase (DNA polymerase that carries out replication of genome) = DNA POL III  In vitro – most active DNA polymerase is DNA POL I: responsible for DNA repair and RNA primer removal  DNA POL II not required for DNA replication o Deletion pol II mutants are still viable  DNA POL IV and V are low fidelity polymerases that lack 3’ to 5’ exonuclease function  DNA POL III Holoenzyme  Holoenzyme = multiprotein complex in which a core enzyme activity is associated with additional components that enhance function  Has three copies of the core DNA POL III enzyme (each comprised of three subunits) and one copy of the five-subunit sliding clamp loader  The sliding clamp loader includes three copies of the tau protein each of which binds one DNA POL III core enzyme

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3 POL III cores o 3 subunits 2 ring shaped beta sliding clamps (improve processivity) 1 clamp loader (assembles beta clamps onto DNA) o Clamp loader has 3 tau subunits that protrude from clamp loader and bind to POL III cores o Total 5 subunits

 Mechanism of DNA Replication 1. Initiation/ Strand Separation 2. Priming the template 3. Loading the Clamp 4. Elongation 5. Termination

6  Replicons  Bacteria: single chromosome, single origin of replication  Replicon: unit of DNA that is replicated / origin where replication initiated and may contain terminus where it stops  Ter = replication fork traps  Basic replication model: binding of the initiator to the replicator stimulates initiation of replication and the duplication of the associated DNA  Simultaneous Replication of both DNA strands: Two replication forks  Two replication forks move around the genome from oriC and meet + finish replication at point halfway around the chromosome from the origin where they run into each other  Either side of meeting point are 23 base pair termination sequences called ter  Ter sequences act as replication fork traps which can stop a replication if it overshoots a little (can happen if one fork stalls)

 Genetic Identification of replicators  A plasmid containing selectable marker is cut with a restriction enzyme that results in the excision of the plasmid’s normal replicator o Leaves DNA fragment that lacks a replicator  To isolate a replicator from a particular organism, DNA is cut with same restriction enzyme then ligated into cut plasmid to re-create circular plasmids – each including a single fragment derived from the test organism  DNA then transformed into host organism and recombinant plasmids are selected using a selectable marker on the plasmid  Isolation of plasmid from host cell and sequencing of inserted DNA allows for the identification of the fragment sequence that contains the replicator  Cells that grow are able to maintain the plasmid and its selectable marker, indicating that the plasmid can replicate in the cell and must contain a replicator  Experimental Demonstration of BIDIRECTIONAL DNA REPLICATION  Bidirectional replication: two replication forks are present which move in opposite directions  Pulses of radioactivity can be used to label the movement of replication forks and distinguish between unidirectional and bidirectional DNA replication  Newly formed replication bubbles initially labelled with low-activity label and later with high activity label o If replication is bidirectional, both sides of the replication eye will have high density label  E. Coli oriC  oriC initiation observed by electron microscopy

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 DNA elements that make up the well-characterized oriC  single replicator required for e. coli chromosomal replication = oriC  identified by recombinant DNA plasmids  245 bp long  Contained 4 copies of a nine nucleotide (9-mer) consensus sequence (called R sites for repeat) to which bacterial initiator protein called DnaA binds  To one side of the DnaA 9 mer sites are 3 A=T rich direct repeats of 13 bp each o = DNA unwinding element, first area in oriC to unwind after initiator binds  Activation of oriC and assembly of bacterial replication fork follows an ordered sequence of events  At least 10 different proteins participate in the initiation phase of replication  Together they open DNA helix at origin and establish a prepriming complex

 Initiation of DNA replication  Alberts et al. have described the collection of proteins – the replisome – at the replication fork as similar to a tiny sewing machine

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Machinery is similar in eukaryotes and prokaryotes but has been best described in e. coli Two types of enzyme are required to convert dsDNA to ss template used in DNA replication o Helicase (DnaB) that separates the strands using ATP hydrolysis o ssDNA binding protein (SSB) that binds to ssDNA and prevents return to a duplex

 Accessory proteins (initiation): DNA Helicases  encoded by DnaB  300 kDa hexameric ring with six identical subunits that bind and hydrolyzes ATP to separate DNA strands  Migrates along the ssDNA (lagging strand template) in the 5’to 3’ direction, unwinding the DNA as it travels  When ATP is added to DNA helicase bound to ssDNA, helicase moves with a defined 5’ to 3’ polarity on the ssDNA o Bound to lagging strand template at replication fork (important when it associates with primase to prime the Okazaki fragments)  DNA helicase pulls ssDNA through a central protein pore Supercoils Revisited  The presence of negative supercoils in DNA may contribute to replication by favouring unwinding of the helix  Helicases unwind DNA at the replication fork without nicking it and the value for Twist decreases; subsequently elsewhere in the DNA compensating positive supercoils appear (which increase writhe)  As positive supercoils accumulate in front of the replication fork, topoisomerases rapidly remove them  The action of topo II (DNA gyrase) removes the positive supercoils induced by a replication fork  Although topo II illustrated in diagram, topo I can also remove positive supercoils generated by a replication fork Single Stranded Binding Proteins  Coat ssDNA  SSB mutants are quick stop (type I)  Bind cooperatively (binding of one promotes binding of another to immediately adjacent ssDNA)  Keeps parental strands separate to act as templates and protects them from endonucleases

9  Accessory proteins (priming) RNA Primase  Primase encoded by dnaG  Smaller than conventional RNA polymerase – makes ~10 base RNA primers  E. coli (and phages) need at least one more protein (DnaB – helicase)  DnaG and DnaB form primosome  Primosome remains with replisome during elongation 1. Primes Okazaki fragments to build lagging strand 2. DnaB helicase moves on lagging strand – keeping primosome there for lagging strand priming

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 Model for initiation of replication in e. coli Major events in the initiation of e. coli DNA replication - (1) Multiple DnaA:ATP proteins bind to the repeated 9-

mer sequences within oriC - HU (histone-like) protein binds to stimulate formation of the replication bubble Binding of DnaA:ATP to these sequences leads to strand separation within 13-mer repeats - Mediated by ssDNA binding protein domain in DnaA:ATP that elongates and changes structure of the associated ssDNA such that it cannot hybridize to the complementary ssDNA - DNA is bent with help of proteins such as HU Priming Preparation - DnaA: the initiator - DnaB: forms hexamer, helicase - Two DnaB hexamers are introduced to origin by interacting with DnaA (one for each strand to expand the bubble bidirectionally) - DnaC: helicase-loading protein; can pry open the hexameric DnaB ring and slip it into the ssDNA at the bubble - Called prepriming complex because the next step is to form the first primer -

a complex between DnaB helicase and the DnaC DNA helicase loader associates with the DNA bound origin

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a ssDNA binding domain in DnaC and protein-protein interaction between DnaA and DnaB/DnaC mediate these interactions

- DNA helicase loader (DnaC) catalyzes the opening of the DNA helicase protein ring and placement of the ring around the ssDNA at the origin

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 How do we know that DnaB helicase moves in the 5’ to 3’ direction?  Different helicases can move in different directions 1. DnaB helicase is added to a long, ssDNA that has short 32-P labelled DNA strands of different sizes anneal to its ends a. Each annealed strand has a short ss tail that mimics a replication fork 2. DnaB initially binds to the ssDNA region, then translocates in one direction to unwind one of the two annealed -DNA duplexes 3. DNA unwinding products are analyzed on a gel Results  DnaB displaces only the 722-mer, revealing that DnaB helicase moves in the 5’ to 3’ direction along the ssDNA  DnaB helicase expands the replication bubble followed by RNA primer synthesis

- The DNA helicases each recruit a primase (DnaG) that synthesizes an RNA primer (~10 bases) on each template - The RNA primer causes the helicase loader to release from the helicase, resulting in the activation of DNA helicase - The movement of the DNA helicases also removes any remaining DnaA bound to the replicator  Synthesis of RNA primer by primases  Primase encoded by dnaG in E. coli  Smaller than conventional RNA polymerase – makes ~10 base RNA primers (11-13 bp in E. Coli)  Primase must bind the DNA helicase for activity (primosome) and this localizes primase action to the replication fork  Lagging strand needs numerous RNA primers, while leading strand has one  A strand that is lagging in one replication fork will be leading in the other  Summary of DNA replication initiation mechanism  E. coli: one origin (250bp, 5x9mer & 3x13mer), two replication forks, one replicon (entire genome)  Eukaryote: many origins, two replication forks/origin, many replicon  Two replication forks move bidirectional (not in all cases)  The initiator (DnaA in E. coli) binds to replication origin (oriC in E. coli), which forms open complex together with ATP, oriC and HU for separating DNA strands at DNA unwinding elements  The subsequent recruiting of two helicases (DnaB) in both strands expands the replication bubble for synthesis of the first RNA primer by primase  DnaB, a hexameric ring moves only in the 5’-3’ direction – separates parental strands. SSB proteins – keep parental strands separate, binds cooperatively to ssDNA  The RNA primer is made by primase (DnaG), forms primosome with DnaB, and recruits DNA Pol III holoenzyme for replication elongation

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 How is re-initiation of DNA replication inhibited?  Regulation occurs at initiation step because once replication has begun, cell is committed to it  Inhibit replication initiation through methylation of oriC by Dam methylase (DNA adenine methyltransferase)  Inactivation of DnaA via ATP hydrolysis by Hda. DnaA-ADP bound to oriC cannot form the open complex  Conversion of DnaA-ADP to DnaA-ATP is slow (~30 mins) Recruiting Pol III holoenzyme Structure of Sliding DNA clamp  Sliding clamp in E. coli is composed of two copies of the beta protein  Slides along DNA and binds to back end of Pol III  Increases speed of replication from 10 bp/s to 1kb/s  Increases the processivity from 1-10 bp to ~100 kb per binding event o Relatively low processivity of DNA pols leads to frequent release from the primer: template junction but the association of the polymerase with the sliding clamp prevents diffusion away from the DNA o After DNA pol has completed synthesis of template, absence of a primer: template junction causes change in DNA pol that releases it form the sliding clamp The clamp loader helps beta sliding clamp assemble onto primed DNA

13  Why do we need three copies of pol III catalytic core + sliding clamp?  Answer: architecture of replisome: holoenzyme + helicase (DnaB) + primase  One is used for replication of leading strand while the other two are used for replication of the lagging strand  HOW DOES REPLICATION OCCUR IN THE OPP DIRECTION IF EVERYTHING IS CONNECTED?  The trombone model of replication fork function

Lagging strand initiation:  After each DNA helicase has moved 1000 bases, a second RNA primer is synthesized on each lagging strand template, and a sliding clamp is loaded  Resulting primer: template junction is recognized by a second DNA pol III core enzyme in each holoenzyme resulting in the initiation of lagging strand synthesis  

Leading-strand synthesis and lagging strand synthesis are now initiated at each replication fork Each replication fork will continue to the end of the template or until it meets another replication fork moving in the opposite direction

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All the players work together at the replication fork Both polymerases can then move together as part of a single replicative complex (replisome) without violating the 5’ → 3' directionality rule for synthesis of a DNA strand DNA polymerase on the lagging strand moves from one completed fragment on the template to a site closer to replication fork Once the polymerase assembling the lagging strand reaches the 5' end of the Okazaki fragment made during the previous round, the lagging-strand template is released – The polymerase then begins work at the 3' end of the next RNA primer toward the fork The trombone model of DNA replication describes the coordinated effort to replicate the leading and lagging stand at the same time

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