Plant Molecular and Cellular BiologyLecture 5: DNA Replicase Structure

& Function

Gary Peter

Learning Objectives

1. List and explain the mechanisms by which eukaryote DNA is replicated

2. Describe and explain the structure and functions of the enzymes and their subunits that replicate DNA in eukaryotes

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 ε 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 polymeraseEach subunit has 3 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).

Dynamics of the Sliding Clamp

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. –DNA dependent hydrolysisB) Pol III is unable to overcome its inherent fidelity to incorporate opposite a damaged base at a lesion in the DNA template, and it stalls. Stalling allows an error-prone polymerase, such as Pol IV that passively travels on β, the time to switch places with Pol III on β and replicate past the lesion. C) Pol III binds tightly to β via the its C terminus. However, when replication is complete, the polymerase must release from β to rebind 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, severing the connection between the polymerase and the clamp

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

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

Assembly of the β Sliding Clamp

The nucleotide free clamp loader has low affinity for the clamp

δ binds tight to β probably sequestering or blocking the other subunits without ATP

When ATP binds the γ-complex changes conformation and δ opens one dimer interface

The clamp-clamp loader complex has a high affinity for primed DNA-templatesThe DNA activates the ATPase activity and allows ring closure and ejection of the loader complexAfter ATP hydrolysis, the clamp loader has a low affinity for DNA until ADP dissociates and the γcomplex binds ATP

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

10-15 bp 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 protein 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

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

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

Dynamic Organization of the Replisome: Lagging Strand Synthesis

Lagging-strand replication is a discontinuous and starts that repeats every 1–3 s. Each Okazaki fragment is initiated by primase, which synthesizes an RNA primer of about 10–12 nucleotides. Primase action requires interaction with DnaB, which involves a C-terminal region of primase. Primase extends the RNA in the opposite direction of helicase unwinding and is presumed to separate from DnaB, which may account for its observed distributive action. Primase remains attached to the RNA primed site through its interaction with SSB. Although primase eventually dissociates, release of primase is accelerated by the χ subunit of the clamp loader, which binds SSB in a competitive fashion, recruiting the clamp loader to the DNA template to compete with primase. The clamp loader then places β onto the primer for the lagging-strand polymerase.As the lagging polymerase extends a fragment, a loop is generated because it is connected to the leading polymerase (via the clamp loader), yet extends DNA in the opposite direction. The 1–3 kb Okazaki fragment will be completed within a few seconds , and at this point, the core must rapidly release from DNA to start the next fragment . The highly processive Pol III requires a specific mechanism for this release step, which disengages core from β, leaving the β clamp behind on the finished fragment. The release step occurs only at a nick, thus ensuring completion of the fragment, and requires the τ subunit . The lagging-strand core is now free to bind a new β clamp placed on the next RNA primer by the clamp loader. Johnson & O’Donnell 2005 Ann. Rev. Biochem. 74: 283-315

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

Clamp Protein Conservation

E. Coli β clamp Yeast PCNAJohnson & O’Donnell 2005 Ann. Rev. Biochem. 74: 283-315

Conservation of the Clamp Loader Structure

Structural comparison of the Pfu clamp-loading complex with the E. coli clamp loader {gamma} complex and the yeast clamp loader-clamp binary complex

E. coli

yeast

Pfu

Pfu

Miyata, Tomoko et al. (2005) Proc. Natl. Acad. Sci. USA 102, 13795-13800

Replisomecomponent

Saccharomyces cerevisiae (kDa) Human (kDa) Function and remarks [Schizosaccharomyces pombe name (S.p.)]

RFCa RFC (277.7)a RFC (314.9)a Pentameric clamp loadera

RFC1 (94.9) p140 (128.2) Binds ATP; phosphorylatedRFC2 (39.7) p37 (39.2) Binds ATPRFC3 (38.2) p36 (40.6) Binds ATPRFC4 (36.1) p40 (39.7) Binds ATPRFC5 (39.9) p38 (38.5) Binds ATP or ADP

PCNA PCNA (28.9) PCNA (28.7) 87 kDa homotrimeric processivity sliding clampa

Pol δa Pol δ (220.2)a Pol δ (238.7)a Replicative DNA polymerasea

Pol3 (124.6) p125 (123.6) DNA polymerase, 3'-5' exonuclease, binds PCNA; subunit A (S.p. Pol3)

Pol31 (55.3) p50 (51.3) Structural subunit; subunit B (S.p. Cdc1)

Pol32 (40.3) p66 (51.4) Binds PCNA; subunit C (S.p. Cdc27); binds Pol α large subunit

— p12 (12.4) Structural, stimulates processivity; subunit D (S.p. Cdm1)

Pol εa Pol ε (378.7)a Pol ε (350.3)a Replicative DNA polymerasea

Pol2 (255.7) p261 (261.5) DNA polymerase, 3'-5' exonuclease (S.p. Pol2/cdc20)Dpb2 (78.3) p59 (59.5) Binds polymerase subunit (S.p. Dpb2)

Dpb3 (22.7) p17 (17.0) Binds Dpb4Dpb4 (22.0) p12 (12.3) Present in ISW2/yCHRAC chromatinremodeling complex (S.p. Dpb4)

Pol αa Pol α (355.6)a Pol α (340.6)a DNA polymerase/primasea

Pol1 (166.8) p180 (165.9) DNA polymerasePol12 (78.8) p68 (66.0) Structural subunitPri2 (62.3) p55 (58.8) Interacts tightly with p48Pri1 (47.7) p48 (49.9) RNA primase catalytic subunit

MCMa MCM (605.6)a MCM (535)a Putative 3'-5' replicative helicasea

Mcm2 (98.8) Mcm2 (91.5) Phosphorylated by Dbf4-dependent kinase

Mcm3 (107.5) Mcm3 (91.0) Ubiquitinated, acetylatedMcm4 (105.0) Mcm4 (96.6) Helicase with MCM6,7; phosphorylated by CDK; aka Cdc54Mcm5 (86.4) Mcm5 (82.3) Aka Cdc46; Bob1 is a mutant formMcm6 (113.0) Mcm6 (92.3) Helicase with MCM4,7Mcm7 (94.9) Mcm7 (81.3) Helicase with MCM4,6; ubiquitinated

RPAa RPA (114)a RPA (100.5)a Single-stranded DNA-binding proteina

RPA70 (70.3) RPA70 (70.3) Binds DNA, stimulates Pol αRPA30 (29.9) RPA30 (29) Binds RPA70 and 14, phosphorylated

RPA14 (13.8) RPA14 (13.5) Binds RPA30

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

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

DNA primaseDNA helicaseSingle strand binding proteinsDNA ligaseDNA polymerasesTopoisomerases

Eukaryotic DNA ReplicationMany conserved molecular mechanisms

Two separate DNA polymerases δ/ε for the leading strand and α for the lagging strand

DifferencesMultiple Oris exist that fire in a coordinated way

Euchromatin replicates first

Larger, less well defined orisequences Old nucleosomes stay attached and new nucleosomes are addedPreori complexes stable on DNA through cell cycle –activated by S-cdk