Plant Molecular and Cellular Biology Lecture 4: E. coli DNA Replicase Structure & Function. Gary Peter

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1 Plant Molecular and Cellular Biology Lecture 4: E. coli DNA Replicase Structure & Function Gary Peter

2 Learning Objectives 1. List and explain the mechanisms by which E. coli DNA is replicated 2. Describe and explain the structure and functions of the enzymes and their subunits that replicate DNA in E. coli

3 Processivity Wang et al., Nucleic Acids Res. 2004; 32(3): The number of nucleotides added during each binding and release from the primed template The ability of the DNA polymerase to remain associated with the DNA template Typical processivity of enzymes in vitro Klenow nt T7 300 nt Taq 22 nt Pfu 6.4 nt

4 Strand Displacement Activity Strand Displacement: The ability to displace downstream DNA encountered during synthesis. Protocols such as the isothermal amplification method Strand Displacement Amplification (SDA) exploit this activity. When new synthesis starts at a nick it displaces a strand. The displaced strand then itself becomes a template for the synthesis of a new strand.

Strand Displacement & Processivity of Bacteriophage Phi29 DNA Polymerase This polymerase has excellent strand displacement activity and high processivity and is used in strand displacement

5 Strand Displacement & Processivity of Bacteriophage Phi29 DNA Polymerase This polymerase has excellent strand displacement activity and high processivity and is used in strand displacement amplification (SDA) High displacement is likely due to a tunnel that is too small for dsdna to enter and requires/induces strand separation The high processivity is likely due to topological encirclement of both the downstream template and the upstream dsdna This structure abrogates the need for ancillary factors such as helicase and the clamp Kamtekar et al., 2004 Mol. Cell 16 (4):

6 Functionality of Various DNA Polymerases 3'->5' Proofreading Strand Displacement Primary Applications Mesophilic DNA Polymerases phi29 DNA Polymerase Strand Displacement Applications T4 DNA Polymerase Polishing Ends, 2nd Strand Synthesis DNA Polymerase I ++ -* Nick Translation DNA Polymerase I, Klenow Fragment Polishing Ends Klenow Fragment (3' -> 5' exo-) Labeling T7 DNA Polymerase (unmodified) Site Directed Mutagenesis Terminal Transferase - NA 3' terminal Tailing *Degrades displaced strand e/polymerases/polymerases_from_neb.asp

7 3'->5' Proofreading Strand Displacement Primary Applications Mesophilic DNA Polymerases hermophilic DNA Polymerases Phusion High Fidelity DNA Polymerase PCR (high fidelity) 3'->5' Proofreading Strand Displacement Primary Applications Phusion Hot Start High Fidelity DNA Polymerase DyNAzyme EXT DNA Polymerase Hot Start PCR (high fidelity) PCR (difficult or long) Other Mesophilic Polymerases DNA M-MuLV Reverse Transcriptase cdna Synthesis DyNAzyme II Hot Start DNA Polymerase Taq DNA Polymerase Vent R DNA Polymerase - - PCR (hot start) - -* PCR (routine), Primer Extension PCR (high fidelity), Primer Extension AMV Reverse Transcriptase E. coli Poly(A) Polymerase NA cdna Synthesis 3 labeling of RNA Vent R (exo-) DNA Polymerase Deep Vent R DNA Polymerase Deep Vent R (exo-) DNA Polymerase PCR, Sequencing PCR (high fidelity), Primer Extension PCR (long), Primer Extension 9 N m DNA Polymerase Primer Extension Therminator DNA Polymerase Bst DNA Polymerase, Large Fragment Chain Terminator Applications Strand Displacement Applications

8 Overview of Basic Steps in DNA Replication 1. Unwinding of the DNA strands 2. Recruitment of DNA polymerase complex & auxiliary factors 3. Initiation of new chain 4. Elongation of the new chain by addition of mononucleotides 5. Covalent closure of the new chains to form one new DNA molecule

9 Standard Biochemical Approach to Identify and Characterize the Proteins/Enzymes that Mediate a Specific Process Identify proteins involved Determine the stiochiometry of the subunits Determine the structure and function(s) of the subunits Determine the spatial arrangement of the subunits Determine the dynamics and steps in the reaction each one mediates Determine the regulation

10 Prokaryotic Replication Fork Leading strand (5 >3 ) Lagging strand (3 >5 ) Enzymes DNA primase DNA helicase Single strand binding proteins DNA ligase DNA polymerases Topoisomerases

11 Replisome

12 Close Association of Proteins into a Replisome at the Fork DNA polymerase III holocomplex Primosome DNA helicase and DNA primase located at the center of the fork where the two strands of the helix are unwinding bound to DNA pol III

13 Model for the Spatial Organization of the the Replisome 2003 Molecular Microbiology, 49,

14 DNA Polymerase III - Holoenzyme A holoenzyme is the fully functional form of an enzyme which contains all of the necessary subunits to be fully active DNA Polymerase Holoenzyme Core enzyme The sliding clamp Clamp loading complex

15 Comparison of DNA polymerases I and III Structure Activities DNA polymerase III DNA Pol III holoenzyme is an asymmetric dimer; i. e., two cores with other accessory subunits. It can thus move with the fork and make both leading and lagging strands. Polymerization and 3'-to-5' exonuclease, but on different subunits. This is the replicative polymerase in the cell. Can only isolate conditional-lethal dnae mutants. Synthesizes both leading and lagging strands. No 5' to 3' exonuclease activity. DNA polymerase I DNA Pol I is a monomeric protein with three active sites. It is distributive, so having 5'-to-3' exonuclease and polymerase on the same molecule for removing RNA primers is effective and efficient. Polymerization, 3'-to-5' exonuclease, and 5'-to-3' exonuclease (mutants lacking this essential activity are not viable). Primary function is to remove RNA primers on the lagging strand, and fill-in the resulting gaps. Vmax (nuc./sec) 250-1,000 nucleotides/second. This is as fast as the rate of replication measured in Cairns' experiments. Only this polymerase is fast enough to be the main replicative enzyme. 20 nucleotides/second. This is NOT fast enough to be the main replicative enzyme, but is capable of "filling in" DNA to replace the short (about 10 nucleotides) RNA primers on Okazaki fragments. Processivity Molecules/cell Highly processive. The beta subunit is a sliding clamp. The holoenzyme remains associated with the fork until replication terminates molecules/cell. In rapidly growing cells, there are 6 forks. If one processive holoenzyme (two cores) is at each fork, then only 12 core polymerases are needed for replication. Distributive. Pol I does NOT remain associated with the lagging strand, but disassociates after each RNA primer is removed. About 400 molecules/cell. It is distributive, so the higher concentration means that it can reassociate with the lagging strand easily.

16 DNA Polymerase III Core Enzyme Structure A heterotrimer of the 3 subunits with different functions in a 2:2:2 stiochiometry α subunit is the DNA polymerase with sequence similarity to C family polymerases No crystal structure exists for this polymerase

17 DNA Polymerase III Core Enzyme Function The core complex can catalyze DNA synthesis (20 nt/s) Without ε subunit the enzyme is not highly processive 1500 nt with each binding and release Presence of e stimulates processivity this helps insure the fidelity as higher rates of DNA synthesis have the proofreading activity Subunit α ε θ Function 5-3 DNA polymerase activity- no proofreading activity (8 nt/s) 3-5 proofreading exonucelase activity Stimulates proofreading exonuclease (not an essential gene)

18 DNA Polymerase III β sliding clamp: Structure Interacts with the α subunit of the DNA polymerase 3 domains Assembles into a dimer with a circular structure and 35 angstrom diameter hole in the middle where DNA is bound

19 Sliding Clamp of DNA Polymerase: Function Increases the rate of DNA synthesis (750 ntd/s) Confers extended processivity to the DNA polymerase (>50 kb).

20 a) The γ complex clamp loader associates tightly with β when bound to ATP. DNA triggers ATP hydrolysis, resulting in low affinity for β and DNA. (b) When Pol III, the replicative polymerase, encounters a lesion in the DNA template, it stalls, unable to overcome its inherent fidelity to incorporate opposite a damaged base. Stalling allows an error-prone polymerase, such as Pol IV (red) passively traveling on β, an opportunity to trade places with Pol III on β to replicate past the lesion. [Adapted with permission from (135).] (c) Pol III maintains a tight grip on β via the polymerase C terminus. However, when it completely replicates its substrate DNA, the polymerase must release from β to recycle to the next primed site. The τ subunit modulates this interaction, binding the polymerase C tail only when no more singlestranded template is present. This severs the connection between the polymerase and the clamp

21 DNA Polymerase III The Clamp loading Complex Structure The clamp loader is composed of 5 subunits that are essential for its function and 2 subunits that link it to SSB and primase Johnson & O Donnell 2005 Ann. Rev. Biochem. 74:

22 DNA Polymerase III The Clamp loading Complex Function The γ complex uses the energy of ATP binding and hydrolysis to topologically link β to a primed DNA, then it ejects from DNA, leaving the closed clamp behind. The three γ subunits bind ATP and are the "motor" of the complex. The δ subunit is the "wrench" because it is the main β clamp-interacting subunit, and it can open the dimer interface by itself. The δ' subunit modulates δ-β contact and is a rigid protein, which remains stationary while other parts move. The χ and ψ subunits are not essential for the clamp-loading mechanism, but χ links the clamp loader to SSB and primase ψ connects χ and strengthens the γ 3 δδ' complex

23 DNA Primase Function & Activity De novo 5 >3 synthesis of short,~10 nucleotide long RNA strands Leading strand synthesis only one RNA primer Lagging strand synthesis RNA primer laid down every ~ nucleotides

24 DNA Primase: Structure There are three functional domains in the protein. The N-terminal 12 KDa fragment contains a zinc-binding motif. The central fragment of 37 KDa contains a number of conserved sequence motifs that are characteristic of primases, including the socalled "RNA polymerase (RNAP)-basic" motif that shows homology with equivalent motifs in prokaryotic and eukaryotic RNAP large subunits. This suggests that primases might share a common structural mechanism with RNAP. The C-terminal domain of approximately 150 residues is the part of the protein responsible for interaction with the replicative helicase, DnaB, at the replication fork.

25 DNA Helicases: Function & Activities Unwinding the dsdna at the replication fork for DNA replication, transcription, repair, recombination ATPase activity used for DNA strand unwinding and movement along single stranded DNA Two different helicases with the ability to move in opposite directions (5 >3 & 3 >5 ) ATP hydrolysis is stimulated by single stranded DNA Helicases move at rates up to 1000 nucleotides/sec

26 DNA Helicase: Structure Hexameric structure with 6 identical subunits Loading onto DNA occurs through the help of loading proteins which promote assembly of the hexamers around the DNA

27 Leading vs. Lagging Strand Synthesis Leading Highly processive Polymerase moves 5-3 Strand displacement is due to the joint action of polymerase III, rep protein and HDP Lagging Short fragments Polymerase moves 3-5 Primase to polymerase switching occurs rapidly Single stranded binding proteins more important DNA polymerase I involvement Elevated DNA ligase involvement

28 Single Stranded Binding Proteins: Function & Activities Involved with DNA replication, recombination, repair Stabilizes ssdna upon binding to the single strands after the helix is opened by helicases

concentrations (<10 mm NaCl and high protein to DNA ratios, Eco SSB displays unlimited cooperative binding to long ssdna, resulting in the formation of

29 Single Stranded Binding Proteins Structure of E. coli SSB Stable tetrameric organization DNA binding domain makes extensive contacts with ssdna Two forms of cooperative binding At low monovalent salt concentrations (<10 mm NaCl and high protein to DNA ratios, Eco SSB displays unlimited cooperative binding to long ssdna, resulting in the formation of long protein clusters. However, at high salt concentrations (> 0.2 M NaCl or > 3 mm MgCl2 and low protein binding density, Eco SSB binds to single stranded polynucleotides in a limited cooperativity mode, in which the protein does not form long clusters along the ssdna

30 Lagging Strand Synthesis Replication Fork DNA polymerase III Primosome SSB Rnase H DNA polymerase I DNA ligase

31 The primase-to-polymerase switch during lagging strand synthesis (A) DnaB helicase encircles the lagging strand and primase has synthesized a primer. The holoenzyme consists of a dimer of tau that binds two polymerase cores, one gamma complex clamp loader, and two beta clamps. Tau and primase interact with DnaB. Primase must contact SSB to remain on the RNA primer. (B) The chi subunit of gamma complex interacts with SSB, severing the primase-ssb contact and resulting in primase displacement. (C) Primase is then free to synthesize another RNA primer upon contact with DnaB. (B) also shows that the lagging strand polymerase releases the beta clamp and DNA upon finishing an Okazaki fragment. (C) shows that after gamma complex assembles the new beta clamp on the upstream primer, the lagging polymerase recruits the new beta clamp (shaded dark) assembled on the upstream RNA primer for the next Okazaki fragment.

32 The Winding Problem The parental DNA winds tightly ahead of the replication fork In E. coli the replication fork travels at 500 bp/sec Every 10 bp replicated is 1 turn of the DNA helix and the helix ahead of the fork becomes wound tighter (48 revolutions/sec) Solution is provided by DNA topoisomerases These enzymes release the tightly wound DNA They can also release the two new DNAs after replication is completed

DNA Topoisomerase I Produces a transient

rotate freely thereby releasing the tension

33 DNA Topoisomerase I Produces a transient single stranded break in the phosphodiester backbone that allows the two sections of the DNA helix on each side of the break to rotate freely thereby releasing the tension built up from unwinding PNAS :

34 DNA Topoisomerase II

35 Summary The enzymes that conduct DNA replication in E. coli are organized into a replisome that contains two copies of DNA polyermase III which act in concert synthesizing the new strands on both the leading and lagging strands Leading strand synthesis occurs very processively, in contrast lagging strand synthesis involves multiple short strand synthesis and the involvement of DNA polymerase I, SSB, primase and DNA ligase more prominently DNA helicase unwinds the duplex ahead of the replication fork and DNA topoisomerases relieve the supercoiling tension introduced by helicase

DNA Replication II Biochemistry 302. January 25, 2006

DNA Replication II Biochemistry 302 January 25, 2006 Following in Dad s footsteps Original A. Kornberg E. coli DNA Pol I is a lousy replicative enzyme. 400 molecules/cell but ~2 replication forks/cell