Plant Molecular and Cellular Biology Lecture 4: E. …sfrc.ifas.ufl.edu/courses/PCB5530/Lectures/080818.PMCB...Structure DNA Pol III holoenzyme is an asymmetric dimer; i. e., two cores

Plant Molecular and Cellular BiologyLecture 4: E. coli DNA Replicase

Structure & Function

Gary Peter

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

Processivity

The number of nucleotides added during each binding and release from the primed templateThe ability of the DNA polymerase to remain associated with the DNA template

Typical processivity of enzymes in vitro

Klenow 50-60 ntT7 300 ntTaq 22 ntPfu 6.4 nt

Wang et al., Nucleic Acids Res. 2004; 32(3): 1197–1207

Strand Displacement ActivityStrand Displacement:

The ability to displace downstream DNA encountered during synthesis.

Protocols such as the isothermal amplificationmethod 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 BacteriophagePhi29 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): 609-618

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 http://www.neb.com/nebecomm/tech_reference/polymerases/polymerases_from_neb.asp

3'->5'Proofreading

StrandDisplacement

Primary Applications

Mesophilic DNA Polymerases

3'->5'Proofreading

StrandDisplacement

Primary Applications

Mesophilic DNA Polymeraseshermophilic DNA Polymerases

Phusion™ High Fidelity DNA Polymerase

+++++ - PCR (high fidelity)

Phusion™ Hot Start High Fidelity DNA Polymerase

+++++ - Hot Start PCR (high fidelity)

DyNAzyme™ EXT DNA Polymerase + + PCR (difficult or

long)

DyNAzyme™ II Hot Start DNA Polymerase

- - PCR (hot start)

Taq DNA Polymerase - -* PCR (routine),

Primer Extension

VentR DNA Polymerase +++ ++

PCR (high fidelity), Primer Extension

VentR (exo-) DNA Polymerase - +++ PCR,

Sequencing

Deep VentR DNA Polymerase +++ ++

PCR (high fidelity), Primer Extension

Deep VentR (exo-) DNA Polymerase - +++ PCR (long),

Primer Extension

9°Nm DNA Polymerase + +++ Primer Extension

Therminator DNA Polymerase - + Chain Terminator

Applications

Bst DNA Polymerase, Large Fragment

- +++Strand Displacement Applications

Other Polymerases

M-MuLV Reverse Transcriptase

- +++ cDNA Synthesis

AMV Reverse Transcriptase

- +++ cDNA Synthesis

E. coliPoly(A) Polymerase

- NA 3´ labeling of RNA

Overview of Basic Steps in DNA Replication

1. Unwinding of the DNA strands2. Recruitment of DNA polymerase complex &

auxiliary factors3. Initiation of new chain4. Elongation of the new chain by addition of

mononucleotides5. Covalent closure of the new chains to form

one new DNA molecule

Standard Biochemical Approach to Identify and Characterize the Proteins/Enzymes that Mediate a Specific Process

Identify proteins involvedDetermine the stiochiometry of the subunitsDetermine the structure and function(s) of the subunitsDetermine the spatial arrangement of the subunitsDetermine the dynamics and steps in the reaction each one mediatesDetermine the regulation

Prokaryotic Replication ForkLeading strand (5’>3’)Lagging strand (3’>5’)Enzymes

DNA primaseDNA helicaseSingle strand binding proteinsDNA ligaseDNA polymerasesTopoisomerases

Replisome

Close Association of Proteins into a Replisome at the Fork

DNA polymerase III holocomplexPrimosome

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

Model for the Spatial Organization of the the Replisome

2003 Molecular Microbiology, 49, 1157–1165

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 enzymeThe sliding clampClamp loading complex

Comparison of DNA polymerases I and III

DNA polymerase III DNA polymerase I

Structure

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.

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.

Activities

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.

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.

ProcessivityHighly processive. The beta subunit is a sliding clamp. The holoenzyme remains associated with the fork until replication terminates.

Distributive. Pol I does NOT remain associated with the lagging strand, but disassociates after each RNA primer is removed.

Molecules/cell

10-20 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.

About 400 molecules/cell. It is distributive, so the higher concentration means that it can reassociatewith the lagging strand easily.

http://oregonstate.edu/instruct/bb492/lectures/DNAII.html

DNA Polymerase III –Core Enzyme Structure

A heterotrimer of the 3 subunits with different functions in a 2:2:2 stiochiometryα subunit is the DNA polymerase with sequence similarity to C family polymerasesNo crystal structure exists for this polymerase

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 Functionα 5’-3’ DNA polymerase activity- no proofreading activity (8 nt/s)ε 3’-5’ proofreading exonucelase activityθ Stimulates proofreading exonuclease (not an essential gene)

DNA Polymerase III –β sliding clamp: Structure

Interacts with the αsubunit of the DNA polymerase3 domainsAssembles into a dimer with a circular structure and 35 angstrom diameter hole in the middle where DNA is bound

Sliding Clamp of DNA Polymerase: Function

Increases the rate of DNA synthesis (750 ntd/s) Confers extended processivity to the DNA polymerase (>50 kb).

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 single-stranded template is present. This severs the connection between the polymerase and the clamp

DNA Polymerase III – The Clamp loading Complex Structure

Johnson & O’Donnell 2005 Ann. Rev. Biochem. 74: 283-315

The clamp loader is composed of 5 subunits that are essential for its function and 2 subunits that link it to SSB and primase

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 γ3δδ' complex

DNA Primase Function & Activity

De novo 5’>3’ synthesis of short,~10 nucleotide long RNA strands

Leading strand synthesis only one RNA primerLagging strand synthesis

RNA primer laid downevery ~ 100-200 nucleotides

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 so-called "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.

DNA Helicases: Function & Activities

Unwinding the dsDNA at the replication fork for DNA replication, transcription, repair, recombinationATPase 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

DNA Helicase: Structure

Hexameric structure with 6 identical subunitsLoading onto DNA occurs through the help of loading proteins which promote assembly of the hexamers around the DNA

Leading vs. Lagging Strand Synthesis

Leading Highly processivePolymerase moves 5’-3’Strand displacement is due to the joint action of polymerase III, rep protein and HDP

LaggingShort fragmentsPolymerase moves 3’-5’Primase to polymerase switching occurs rapidlySingle stranded binding proteins more importantDNA polymerase I involvementElevated DNA ligaseinvolvement

Single Stranded Binding Proteins: Function & Activities

Involved with DNA replication, recombination, repairStabilizes ssDNA upon binding to the single strands after the helix is opened by helicases

Single Stranded Binding Proteins Structure of E. coli SSB

Stable tetrameric organizationDNA binding domain makes extensive contacts with ssDNATwo 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

Lagging Strand Synthesis

Replication ForkDNA polymerase IIIPrimosomeSSB

Rnase HDNA polymerase I DNA ligase

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 dimerof 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.

http://oregonstate.edu/instruct/bb492/figletters/FigU.html

The Winding ProblemThe 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 DNAThey can also release the two new DNAs after replication is completed

DNA Topoisomerase IProduces 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 2003 100: 10629–10634

DNA Topoisomerase II

SummaryThe 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 strandsLeading 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 prominentlyDNA helicase unwinds the duplex ahead of the replication fork and DNA topoisomerases relieve the supercoiling tension introduced by helicase