DNA Replication IBiochemistry 302January 23, 2006Watson Crick prediction:Each stand of parent DNA serves as a template for synthesis of a new complementary daughter strandFig. 4.12Primer strandTemplate strandProof of semiconservative DNA replicationMatthew Meselson-Franklin Stahl, 1958heavylightFig. 4.14hybrid¾L¼HVisualization of bidirectional replicationJohn Cairns: 3H-thymidine labeling and visualization of 1.7mm E. coli chromosome (note loops → two new daughter DNAs).Autoradiograms of E. coli chromosome subjected to transient pulse 3H-thymidine labeling implied bidirectional replication.Lehninger Principles of Biochemistry, 4th ed., Ch 25Termination and re-initiation occur on opposite sides of E. coli chromosomeFig. 24.12Higher specific activity [3H]-thymidine added at end of replication cycle.Note higher grain density at points of termination and re-initiation.Ross Inman and coworkers: replication initiates at unique origins• Denaturing mapping studies• Bacteriophage λ ~48.5 kbpchromosome partially denatured then fixed to prevent renaturation.• Observed pattern of ssDNA bubbles via EM.• Sequences rich in A=T base pairs served as points of origin in replicating λ.• Confirmed concept of bidirectional replication• oriC subsequently identified in E. colichromosome Lehninger Principles of Biochemistry, 4th ed., Ch 8, 25Basic chemistry of DNA chain elongation•(dNMP)n+ dNTP → (dNMP)n+1+ PPi•Incoming dNTP is positioned in active site by base-pairing w/ template nucleotide.• Polymerase catalyzes reaction between the terminal 3′ OH on the primer strand & the 5′ α phosphorus of dNTP to form 3′− 5′ PDE bond.• Release & hydrolysis of PPiby pyrophosphatase (~19 kJ/mol generated) favors formation of products.• Consequently, chain growth can only occur in one direction.Fig. 24.2Phosphoryl group transfer reactionBasic features of DNA replication (constraints: direction & anti-parallel polarity)• Semi-conservative• Ordered & sequential– Starts at a fixed point– Synthesis in 5′→3′ direction (topological problem) • Semi-discontinuous– Leading stand (same direction as fork)– Lagging strand (opposite fork movement)• Extremely accurate• Activated substrates (dNTPs) Okazaki fragments: In bacteria, ~1000 to 2000 nucleotides long; in eukaryotes, ~150 to 200 nucleotides long.Lehninger Principles of Biochemistry, 4th ed., Ch 25Directionality of DNA synthesis: a very simple model of a replication fork DNA polymerase III, replicates as leading and lagging strand dimerLeading strandLagging strandRNA primerprimasomeFork movementLagging strand synthesis requires repeated RNA priming due to physical constraint imposed by PolIII dimerization at the fork.Fig. 24.1Features of E. coli DNA replication coordination of enzyme activities• Multi-enzyme “replisome”– Substrates: dNTPs (also need primer-template)– Enzymes: Polymerases (PolIII for rapid synthesis & Pol I for editing), DNA gyrase, helicase, primase, & ligase • Three main phases:– Initiation: formation of specific protein:DNA complex at oriC– Elongation: the actual copying process (Figure)– Termination: separation of daughter chromosomes at Ter sites by Tus proteinSSB plays a unique, nonenzymatic role.Lehninger Principles of Biochemistry, 4th ed., Ch 25Enzymes and other proteins involved in DNA replication(some multi-subunit complexes)• DNA polymerase(s)• ssDNA-binding protein (SSB)• Helicase (DnaB)• Primase (DnaG)• Topisomerase(s) (DNA gyrase, topo IV)• DNA Ligase• Initiation factors (DnaA, DnaC, HU, Dam methylase)Lehninger Principles of Biochemistry, 4th ed., Ch 25**DNA Pol I: the first polymerase(discovered by Arthur Kornberg)• Three activities: – “slow” polymerization – 3′ to 5′ exonucleolytic proofreading & repair– 5′ to 3′ exonuclease (RNA primer removal, nick translation)• Modular structure• Not processive enough for genome replication • “Cleanup”/repair enzymeKornberg & BakerDNA Pol I structure: 103 kDa peptide. Limited tryptic digestion → small N-term 35 kDa fragment and larger C-term 68 kDa fragment (Klenow, used for DNA synthesis in vitro)NCFig. 24.5DNA polymerase I: 5′→3′ exo activity NickTranslation −concerted rNMPremoval from 5′end of RNA primer& dNMP addition to 3′ end of Okazaki fragmentDNA polymerase I (and Pol III): 3′→5′proofreading activity• Mechanism based on structural studies• 3′→5′ exonuclease site positioned ahead of the polymerase active site• Mismatch impedes enzyme translocation• Enzyme stalls allowing access to exonuclease active site• Polymerization can then resume in 5′→3′direction.Lehninger Principles of Biochemistry, 4th ed., Ch 25iminoaminoKlenow family of DNA polymerases (fingers, palm, thumb subdomains)Bacteriophage RB69, a cousin of phageT4, 1stoneBacteriophage T7 DNA PolDoublie et al. (1998) Nature 391:251Fig.24.17Molecular basis of primer-template recognition and proofreadingExtensive contacts between the T7 DNA Pol enzyme and DNA minor groove explains Pol DNA-binding w/o sequence specificity, detection of base mis-incorporation.O2N3O2of Pyr and N3 of Pur are universal H-bond acceptors of the minor groove. A-form helix has wider minor groove.Arg429 and Gln615 are strictly conserved.floor of active siteDoublie et al. (1998) Nature 391:251DNA polymerase active site appears capable of opening and closing O Helix = dynamic finger subdomainDoublie et al. (1998) Nature 391:251530530Two metal ion mechanism of catalysis by DNA polymerases (T. Steitz model)Doublie et al. Nature 1998Metal ions (Mg2+) stabilize a multivalent transition state between phosphate oxygens and two conserved Asp residues which 1) polarizes the primer 3′ OH and 2) facilitates the leaving of pyrophosphate.3′E. coli KlenowT7 DNA Polwater5′Fig. 24.18T7 Pol*=