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Chapter 28

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McMaster University
Margaret Fahnestock

Biochem 2B03 Jasmyn Lee Chapter 28: DNA Metabolism: Replication, Recombination and Repair 28.1 How is DNA Replicated?  Rule – identify the 5’ ends and 3’ ends  Rule – biosynthesis of nucleic acids proceeds in the 5’ to 3’ direction  DNA is replicated by a “nucleophilic substitution” reaction o (DNA) + dNTP  (DNA) + PP n n+1 i o Nucleophilic attack of 3’ hydroxyl on the 5’ α phosphate of the incoming NTP  DNA replication is a chemical reaction o O atom in the hydroxyl is electron rich (nucleophile) o P atom in the 5’ α phosphate is electron poor (electrophile) o Inorganic pyrophosphate is displaced from the incoming dNTP o It is a “leaving group”  Many reactions that involve the liberation of PPi are energetically unfavorable – that is oG ’ of the reactants is similar to or less than that of the products o In chemical terms – it is hard for this reaction to move in the “forward” direction  so how is biosynthesis possible?  The key is the pyrophosphate o (DNA) n dNTP  (DNA) n+1 PP i  The enzyme inorganic pyrophosphatase degrades PPi and the removal of this product is what causes the reaction to proceed (think le Chatelier) o Eg/ (DNA) n dNTP  (DNA) n+1 PP i PPi -Inorganic pyrophosphat--> 2P  Removing one product makes it impossible for the reaction to go backwards  Since the pyrophosphate reaction is energetically favorable, the overall reaction is energetically favourable  DNA synthesis does not occur spontaneously  It must be template by an unwound DNA duplex and catalyzed by a DNA-dependent DNA polymerase  DNA Replication is Bidirectional - DNA is unwound locally, and both strands are duplicated o Replication begins at the origin(s) of replication and proceed in both directions from the origin o Bidirectional replication involves two replication forks that move in opposite directions o Demonstrated by John Cairns in 1963 o This raises a serious problem:  Think about what happens as you bidirectionally replicate the DNA duplex to the right – think of the 5’ and 3’ ends o One of the new strands is extending 5’-3’ towards the site where the DNA is being unwound o The other is pointed in the opposite direction  Replication requires unwinding of the DNA helix o Double helix must a) Rotate about its axis while the ends of its strands are held fixed b) Positive supercoils must be introduced, one for each turn of the helix unwound  If the chromosome is circular – DNA would become too tightly supercoiled to allow unwinding of strands  only option b o DNA Gyrase – Type II topoisomerase; acts to overcome the torsional stress imposed upon unwinding; introduces negative supercoils at the expense of ATP hydrolysis 1 Biochem 2B03 Jasmyn Lee  Alter the linking number of dsDNA through phosphodiester bond breakage and reunion o Helicase – drives unwinding reaction; class of proteins that catalyze the ATP-dependent unwinding of DNA double helix  Disrupt the hydrogen bonds that hold the two strands of duplex DNA together  Requires a single stranded region for binding; moves along single strand, unwinding the double stranded DNA in ATP-dependent process o Single Stranded DNA-Binding Protein (SSB) – binds to the unwound strands, preventing their re- annealing  DNA replication is semi discontinuous o DNA Polymerase – carries out DNA replication; uses ssDNA as a template and makes a complementary strand by polymerizing deoxynucleotides in the order specified by their base pairing with bases in the template  Synthesizes DNA in 5’3’ direction, reading the antiparallel template strand in a 3’5’ direction o Leading Strand – 3’5’ strand; opened in the 5’ to 3’ direction during replication which can therefore be replicated continuously o Lagging Strand – 5’3’; opened in the 3’ to 5’ direction during replication and can only be replicated discontinuously for form Okazaki fragments; a sufficient stretch of its sequence has to be exposed for DNA polymerase to read in the 3’5’ sense  Okazaki Fragments – discontinuous stretches of replicated DNA that when joined form the lagging strand; 1000-2000 nucleotides in length o This partly explain the Cairns result and is consistent with the anti-parallel nature of DNA – but also raises some more problems o The leading strand and lagging strand are coordinated – how?  Clearly the biochemistry of DNA synthesis is complex o Historically, the most important work involved genetic experiments (the isolation of bacterial strains with mutations in genes for DNA synthesis) and the biochemical reconstitution of DNA synthesis in a test tube 28.2 What Are The Properties of DNA Polymerase?  DNA Polymerase – enzymes that replicate DNA 1. The incoming base is selected within the DNA polymerase active site 2. Chain growth is in the 5’3’ direction and is antiparallel to the template strand 3. DNA polymerases cannot initiate DNA synthesis de novo – all require a primer oligonucleotide with a 2 Biochem 2B03 Jasmyn Lee free 3’-OH to build upon  Most bacteria (eg/ E. coli) have three DNA polymerase enzymes  DNA Polymerase I – catalyzed the synthesis of DNA in vitro if provided with all four deoxynucleoside-5’- triphosphates (dNTP  dATP, dGTP, dCTP, dTTP), a template DNA strand to copy and a primer o Primer is essential – DNA polymerase can elongate only preexisting chains; cannot join two deosyribonucleoside-5’-triphosphates together to make the initial phosphodiester bond o Primer base pairs with the template DNA, forming a short double stranded region o Primer must posses a free 3’-OH end to which an incoming deoxynucleoside monophosphate is added o A dNTP is selected as a substrate (based on base pairing), pyrophosphate iPP) is released, a the dNMP is linked to the 3’-OH of the primer chain through formation of a phosphoester bond o Modestly processive – synthesizes complementary strand of 3-200 bases  Turnover Number/Processivity – the length of DNA the purified enzyme can produce in vitro before it falls off the template DNA o Differs markedly between the three enzymes – consistent with their biological roles  Pol III is the most processive o Turnover number is >1000 (pol I and II are 20-40) o Pol III – replicates the entire chromosome  A single enzyme can replicate around the entire bacterial chromosome in one continuous reaction o Rate of DNA polymerization is ~1000 bp/second  In contrast – pol I and pol II are involved in DNA repair  DNA polymerase enzymes have exonuclease activities o 3’  5’ exonuclease activity – for removing incorrectly inserted bases  Removes nucleotides from the 3’-end of the growing chain; negates the action of the polymerase activity  Polymerase cannot elongate an improperly base-paired primer terminus; 3’-exonuclease has time to act and remove mispaired nucleotide  3’  5’ exonuclease is slow o 5’  3’ polymerase activity is very slow following a mis- incorporated base  5’  3’ polymerase activity is very fast  Acts upon duplex DNA, degrading it from the 5’end by releasing mononucleotides and oligonucleotides  Can remove distorted (mispaired) segments lying in the path of the advancing polymerase  Plays an important role in primer removal  Nick Translation – DNA polymerase I binds at nicks (single stranded breaks) in dsDNA and move in the 5’3’ direction, removing successive nucleotides o When a base is inserted incorrectly – the enzyme pauses, permitting the exonuclease activity to remove 3 Biochem 2B03 Jasmyn Lee the base so that it can then be removed and the mistake corrected  Arthur Kornberg – Nobel Prize, 1959  DNA replication in vitro o E. Coli extract – all proteins from the cell, dATP, dGTP, dCTP, dTTP, radioactive dTTP, buffer, ATP, CTP, UTP, GTP, Template DNA (usually a bacteriophage chromosome) o The radioactively labeled dTTP is converted from nucleotide to DNA (insoluble in ethanol at low pH) – know DNA synthesis has taken place o E. Coli cells contain between 2000-3000 proteins (at varying levels) o Column Chromatography – identify protein that synthesizes DNA  Separation by charge – ion exchange chromatography o Separation by size – gel filtration chromatography E. Coli DNA Polymerase III Holoenzyme Replicates the E. coli Chromosome  The purified subunits of pol III can be reconstituted in different ways o Note – pol III core is a monomer, pol III is a diner 4 Biochem 2B03 Jasmyn Lee  Why is pol III holoenzyme so processive o (αεθ)2 2ϒδδ’χψβ o β = sliding clamp; tethers core polymerase to the template, accounting for the great Processivity of the DNA polymerase holoenzyme o γ complex = clamp loader; catalyze the ATP-dependent transfer of a pair of β-subunits to each strand of the DNA template o τ complex = processivity switch; on lagging strand, releases DNA template when synthesis of Okazaki fragment is completed and rejoins template at next RNA primer to begin synthesis of next Okazaki fragment  Usually “off”; turns “on” when on lagging strand and when synthesis of an Okazaki fragment is completed  Ejects the β-sliding clamp bound to the lagging strand core polymerase  Almost immediately, the lagging strand core polymerase is reloaded on a new β-sliding clamp at the 3’ end of the next RNA primer and synthesis of new Okazaki fragment commences  DNA polymerase enzyme can’t do “first bond synthesis” o (DNA) n dNTP  (DNA) n+1+ PPi o n > 1 o Pol III solves this problem by using a “primase” to synthesize a short stretch of RNA, known as a “primer” o In E. coli the primase is the DnaG protein  Helicase (DnaB protein) and DNA gyrase (topoisomerase) – opens the DNA in advance of Pol III 5 Biochem 2B03 Jasmyn Lee  SSB (single strand binding protein) – keeps single stranded DNA from annealing  Primase (DnaG) – synthesizes an RNA primer on the lagging strand (the leading strand was primed when replication was initiated)  Lagging strand is looped around; DNA polymerase moves 5’3’ relative to its strand, copying template and synthesizing new DNA strand  β-clamp keeps leading strand replication from falling off DNA  γ-complex loads β clamp on lagging strand  Polymerase I – simultaneously removes RNA primers (5’3’ exo) and extends previous new strand – “nick translation”  DNA Ligase – seals adjacent new Okazaki fragments Proteins Involved in DNA Replication Protein Function DNA Gyrase Unwinding DNA SSB Keeps ssDNA from annealing DnaA Initiation factor; origin-binding protein DnaB 5’3’ helicase (DNA unwinding) DnaC DnaB chaperone; loading DnaB on DNA Primase (DnaG) Synthesis of RNA primer DNA Polymerase III Elongation (DNA synthesis) Holoenzyme DNA Polymerase I Excises RNA primer, fills in with DNA DNA Ligase Covalently links Okazaki fragments Tus Termination Biochemists purified Pol III – genetics isolated Pol III mutations  Biochemistry revealed lots of subunits; genetics revealed lots of genes  Bacterial genetics convention o Gene = dnaA o Gene product (protein) = DnaA o DnaA – initiation factor; binds “origin of replication”; binds and hydrolyzes ATP o DnaB – DNA helicase o DnaC – represses DnaB helicase activity o DnaG – primase; adds RNA primers to initiate synthesis of a new DNA strand  Roles of DnaA, B, C and G proteins are consistent with “slow stop” phenotype of dnaA, dnaB, dnaC and dnaG mutations 6 Biochem 2B03 Jasmyn Lee How does DNA synthesis initiate?  Starts at a specific sequence called the “origin of replication”  Recognized by DnaA protein  What happens during initiation? – bacteria replicating their DNA 1. DnaA binds oriC – does not require ATP 2. Unwinds AT-rich sequence – requires ATP hydrolysis 3. Formation of “pre-priming complex”: DnaA recruits DnaB/DnaC complex – positioned at each new replication fork at edge of unwound region 4. DnaC dissociates from DNAB (helicase activated) 5. DnaB recruits DnaG 6. Synthesis of first primers and assembly of Pol III 28.3 What Are There So Many DNA Polymerases 28.4 How is DNA Replicated in Eukaryotes? Eukaryotes – similar but more complicated replication mechanism  Multiple chromosomes  Chromosomes are bigger (smallest human chromosome is 10x larger than the E. coli chromosome)  Chromosomes have multiple origins  Coordinated action of all origins – they all “fire” at the same time The Eukaryotic Cell Cycle  Four distinct phases o M – mitosis o G1– rapid growth and metabolic activity o S – DNA replication and growth o G2– growth and preparation for cell division  Synchronizes the biochemical activities of all chromosomes  Chromosomes must be replicated once/cell cycle  Progression through the cell cycle is regulated by cyclins and cyclin-dependent kinases (CDKs) o Cyclins are synthesized during one stage of the cycle and degraded later o Cyclins bind and activate CDKs – CDKs are inactive unless complexed with specific cyclin partner o CDK’s control events at each phase of the cycle by targeting specific proteins for phosphorylation o Destruction of the phase-specific cyclin at the end of the phase inactivated the CDK  Initiation of Replication – divided into two steps o Eukaryotic cells initiate DNA replication at multiple origins; two replication forks arise from each origin and move away from each other in opposite directions o Origin Recognition Complex (ORC) – protein complex that binds to replication origins 1. Licensing – late M/early G1 o Overall: ORC binds origin – recruits helicase (MCM) o ORC binds to origin o Proteins bind to ORC forming a prereplication complex (pre-RC) on the origin of replica1ion (G ) o Yeast Model:  ORC binds to origin  ORC recruits Cdc6, Cdt1 and MCM proteins  Cdc6 and Cdt1 – replicator activator proteins  Cdc6 is degraded following replication initiation  MCM proteins – replication licensing factors; they permit DNA replication to occur  MCM proteins assemble as hexameric helicases that render the chromosomes competent for replication 2. Activation – S phase o S-phase cell cycle kinases phosphorylate additional initiation factors 7 Biochem 2B03 Jasmyn Lee o Phosphorylation of the MCM proteins and binding of Cdc45 activated the helicase activity of MCM o Phosphorylation of Sld2 and Sld3; interact with Dbp11 o Recruitment of Dbp11 – which recruits DNA polymerase to replication origins  Dbp – DNA polymerase binding  Sld – synthetic-lethal with Dpb11 o Trigger bidirectional DNA replication from each origin with two diverging MCM complexes serving as helicases o Each helicase unwinds the duplex DNA to provide single-stranded templates for the DNA polymerases that follow S phase Pre-initiation complex “11-3-2” complex Eukaryotic Cells Contain a Number of Different DNA Polymerases  DNA Polymerase α o Has an associated primase subunit o Functions in the initiation of nuclear DNA replication o Given a template, it not only synthesizes an RNA primer of about 10 nucleotides, but it then adds 20-30 deoxynucleotides to extend the chain in the 5’3’ direction  DNA Polymerase δ o Heterotetrameric enzyme o The principal DNA polymerase in eukaryotic DNA replication o Interacts with proliferating cell nuclear antigens (PCNA) protein when carrying out highly processive DNA synthesis o PCNA – eukaryotic counterpart of E. Coli β-sliding clamp; clamps DNA polymerase δ to DNA  Encircles double helix  Trimer not dimer Bacteria Eukaryotes  β-sliding clamp  “Proliferating cell nuclear antigen” (PCNA) –  Dimer the eukaryotic β-clamp  Trimer  DNA Polymerase ε o Has acidic C-terminal extension lacking in other DNA polymerases; this domain is a sensor for DNA damage checkpoint control, halting DNA replication until the damage is repaired  DNA Polymerase γ o DNA-replicating enzyme of mitochondria 8 Biochem 2B03 Jasmyn Lee  DNA Polymerase β o DNA repair 28.5 How Are The Ends of Chromosomes Replicated? Recall:  DNA is replicated by a “nucleophilic substitution: reaction o (DNA) n dNTP  (DNA) n+1+ PPi o Nucleophilic attack of 3’ hydroxyl on the 5’ α phosphate of the incoming NTP o Inorganic pyrophosphatase cleaves PPi making reaction energetically favorable o PPi--Inorganic pyrophospha-->e 2P The End Problem  First proposed as a problem by Alexei Olovnikov, 1971  Elizabeth Blackburn and Cal Harley  Replicate leading and lagging strands  RNA primers in red, new DNA in green  Remove primers, fill in the gaps (pol I) and ligates together (DNA ligase) 9 Biochem 2B03 Jasmyn Lee  Primers are replaced but leave gaps at the ends of the lagging strands  These gaps cant be filled in without primers  The daughter chromosomes are shorter at the 5’ end of one strand Characterization of the “telomere”  Repeated sequence found at the chromosome ends  Form protective caps 1-12kb pairs long  Necessary for chromosome maintenance and stability  1982 – Szostak and Blackburn – Cell 29:245-55  DNA fragments from Tetrahymena that stabilized linear DNAs in yeast cells o Repeated (50-100x) hexanucleotide sequence: CCCCAA  1988 – Moyzis et al., Proc. Nati. Acad. Sci 85:6622-6  Human telomere cloned and sequenced
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