lecture 19 • dna replication key learning...

Lecture 19 • DNA Replication

Key learning goals:• Understand how Acyclovir and related antiviral drugs

work. Understand their mechanisms of selectivity.• Know what a primer for DNA synthesis is. • Understand the function of 5´-3´ exonucleases in DNA

replication.• Understand the roles in replication of: sliding clamp,

clamp loader, SSB, helicase, topo II/gyrase, primase complex.

• Know what E. coli DNA pol I, II, III do.• Understand what RNAseH does in eukaryotic DNA

synthesis.• Understand the Meselson-Stahl experiment and what

it tells us.

Acyclovir (ACV), an important antiviral (pro)drug

viralDNA

polymerase

ACV → ACV-MP → ACV-DP → ACV-TP →! " # $

chaintermination

• ACV is a key drug used against herpes viruses, cytomegalovirus, etc.

• In infected cells, ACV is activated by phosphorylation, making it a dGTP mimic.

• ACV-TP, the active form, lacks a 3´-OH, so it’s a chain terminator.

• ACV is very e!ective and has low toxicity because it is a much be"er substrate for viral TK (1) and viral DNA polymerase (4) than for human TK or human polymerases.

viral thymidine

kinase GMP kinase

nucleotidediphosphate

kinase

DNA Polymerization requires an initiating primer which can be made from either DNA or RNA

DNA-dependent DNA polymerases cannot extend a DNA chain from nothing – they can only add nucleotides to a free 3´-OH!

Template Product RepresentativeEnzymes

Primer required?

DNA DNA DNA polymerase YES

DNA RNA RNA polymerase NO

RNA DNA

HIV reverse transcriptase

Telomerase

YES

RNA RNAInfluenza virusRNA replicase

NO

DNA Polymerizases require an initiating primer made from either DNA or RNA. RNA polymerases don’t.

Question: What chemical group would you expect to find at the 5´ terminal positon of a typical RNA primer? Why?

DNA replication is semiconservative5´

5´

3´

3´3´ 5´

3´5´

Each daughter strand contains one of the original parent strands and one new strand.

Meselson-Stahl experiment demonstrates semiconservative replication

DNA replication is semiconservative5´

5´

3´

3´3´ 5´

3´5´

direction ofreplication

fork movement

The template strands are antiparallel, and polymerase can only synthesize DNA in one direction (5´→ 3´).

How can both strands be replicated at the same time, and in the same overall direction?

DNA replication: lagging strand is synthesized as a series of short Okazaki fragments

C. Gaps between the Okazaki fragments are sealed by DNA Pol I and DNA ligase.

A. RNA primers are laid down by a special DNA-dependent RNA polymerase called Primase

B. Okazaki fragments are elongated by DNA Pol III.

Division of labor among prokaryotic DNA polymerases

Function of DNA polymerase I in gap repair

In addition to its polymerase function, DNA Pol I has a 5´-3´ exonucleaseactivity that removes RNA and DNA primers

RNA primer

Synthesis of all genomic DNA involves the highly coordinated action of multiple polypeptides. These proteins assemble two new DNA chains at a remarkable pace, approaching 1000 nucleotides (nt) per second in E. coli.

If the DNA duplex were 1 m in diameter, then the following statements would roughly describe E. coli replication. The fork would move at approximately 600 km/hr (375 mph), and the replication machinery would be about the size of a FedEx delivery truck. Replicating the E. coli genome would be a 40 min, 400 km (250 mile) trip for two such machines, which would, on average make an error only once every 170 km (106 miles).

The mechanical prowess of this complex is even more impressive given that it synthesizes two chains simultaneously as it moves. Although one strand is synthesized in the same direction as the fork is moving, the other chain (the lagging strand) is synthesized in a piecemeal fashion (as Okazaki fragments) and in the opposite direction of overall fork movement. As a result, about once a second one delivery person (i.e., polymerase active site) associated with the truck must take a detour, coming o! and then rejoining its template DNA strand, to synthesize the 0.2 km (0.13 mile) fragments.

— Tania Baker and Stephen Bell (1998) Cell 92:295

Eukaryotic primers contain RNA and DNAThey are made & removed in two steps

Note that FEN removes the error-ridden DNA synthesized by Pol (alpha), which doesn’t proofread

Division of labor among eukaryotic DNA polymerases

There are >12 eukaryoticDNA polymerases

Pol (alpha) is a subunit of the primosome

note that it does not proofread)

Pol (delta) isa majorreplicativepolymerasethat probably makes Okazakifragments onthe lagging strand

Pol (epsilon)replicates theleading strand

What you need to remember: The headline for this slide. And: there are a bunch of eukaryotic polymerases that do di!erent things. Distinct polymerases synthesize primers, leading, and lagging strands. The ones that do big-time replication proofread, but some others don’t.

The Pol III holoenzyme is an asymmetric dimer that catalyzes both leading and lagging strand synthesis

Note looping of the lagging strand DNA!

SSB = single-stranded DNA binding protein. SSB keeps melted DNA from re-annealing & protects the exposed bases.

1000 bp/s

100 turns of the helix per second — DNA must unwind at almost 10,000 r.p.m. !!!

The locking carabiner is a processivity factor

(it keeps the climber from falling o! the rope)

The sliding clamp is a processivity factor(it keeps the polymerase complex from falling o! the DNA)

Color-coded by charge density:blue is positive, red is negative.

PDB 2POL

In mammals, the sliding clamp is called PCNA

* For an alterative view, see Lehninger Fig. 25-14.

Replication initiates at a single site, OriC, on the circular E. coli chromosome

replicationforks

In addition to being semiconservative, and discontinuous, DNA replication is bidirectional— two forks diverge from each origin

replication of a circular DNA duplex in a bacterium

Bacterial chromosomes can initiate replication more than once in a cell cycle. Under conditions, each daughter cell already contains a chromosome that has been partially replicated!

oriC(origin of replication)note: each line segment in

this diagram represents a DNA double helix.

(When, how often)

Initiation of DNA replication

Leading

Lagging

(Where)

The circular E. coli chromosomeinitiates replication at a single location.

This DNA sequence is called OriC.

OriC is specifically bound by a set of proteins that melt the DNA and place two “replication factories” on the DNA, facing in opposite directions.

Multicellular eukaryotes face another problem: They usually have a LOT more DNA than bacteria.

Mycoplasma: 500,000 bpSalmonella: 5,000,000 bp

Brewer’s yeast: 12,000,000 bpMalaria parasite: 20,000,000 bp

Drosophila (vinegar fly): 200,000,000 bpSea urchin: 800,000,000 bp

Human: 3,000,000,000 bpAmphibian: up to 100,000,000,000 bp

...And eukaryotic DNA polymerization is generally slower than in bacteria!

Solution: use many origins of replication.

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Matreiya Dunham, UW Genome Sciences

Potential originsof replication in brewer’s yeast,Saccharomycescerevisiae

John J. Wyrick, et al.

Genome-Wide Distribution of ORC and MCM Proteins in S. cerevisiae : High-Resolution Mapping of Replication Origins

Science 294, 2357 (2001) DOI: 10.1126/science.1066101

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Updated Meselson-Stahl experiment maps origin locations and firing order

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h"p://en.wikipedia.org/wiki/Cell_cycle

I = Interphase

M = Mitosis

G1 = Gap 1S = SynthesisG2 = Gap 2

{

The eukaryotic cell cycle

*

*

*The first key genes known to control the eukarytic cell cycle were discovered here @ UW by Lee Hartwell (Nobel Prize 2001)

DNA damage checkpoints:* at G1/S* during S phase* at G2/M

*

*

*

The eukaryotic cell cycle

I = Interphase

M = Mitosis

G1 = Gap 1S = SynthesisG2 = Gap 2

{

h"p://en.wikipedia.org/wiki/Cell_cycle

review

EMB O reports V OL 6 | N O 11 | 2005 ©2005 EUROPEA N M OLEC ULAR BIOLO GY ORG A NIZATIO N

The chromosome cycleJ.J. Blow & T.U. Tanaka

1030

These can be grouped into four categories (proteolysis, inhibitoryphosphorylation , nuclear export and inhibition by geminin),which we briefly examine in turn below.

Proteolysis. In yeast, rising C D K levels at the end of G 1 causephosphorylation of Cdc6 (fission yeast Cdc18), which targets itfor ubiquitylation and subsequent proteolysis (Drury et al, 1997;Jallepalli et al, 1997). In metazoans, Cdc6 levels remain relat-ively constant throughout the cell cycle . Instead , metazoan Cdt1is ubiquitylated and degraded at the onset of S phase , partlyowing to C D K-dependent phosphorylation , and this is importantin preventing re-licensing of replicated D N A (N ishitani et al,2001; Li et al, 2003; Zhong et al, 2003; Arias & Walter, 2005; Li& Blow, 2005). In metazoans, O RC and Cdc6 are also regulatedby ubiquitylation and proteolysis, although the physiologicalconsequences of this regulation remain uncertain .

Inhibitory phosphorylation. In yeast, the O rc2 subunit is phos-phorylated by C D Ks during late G 1 , S, G 2 and mitosis, and thishas a role in preventing re-licensing of replicated D N A (Nguyenet al, 2001; Vas et al, 2001). C D K-dependent phosphorylation ofthe mammalian O RC and Cdc6 probably inhibits their binding toD N A during mitosis (Li et al, 2004).

Nuclear export. In budding yeast, but not fission yeast ormetazoans, C D K activity leads to the nuclear export of bothMcm2–7 (Tanaka et al, 1997; Labib et al, 1999) and Cdt1 . Thisprevents these proteins from gaining access to their D N A sub-strate and so prevents re-licensing of replicated D N A . Export ofMcm2–7 and Cdt1 is interdependent, and might be driven byphosphorylation of Mcm2–7 .

Inhibition by geminin. G eminin is a small protein that bindsand inhibits Cdt1 (Mc G arry & Kirschner, 1998; Wohlschlegel et al, 2000; Tada et al, 2001). So far, it has been described only inmetazoans. G eminin is ubiquitylated by the APC/C during latemitosis and early G 1 . In somatic cells, this action targets it forproteolysis; however, in early embryos, ubiquitylation of gemi-nin leads to its inactivation rather than its degradation (H odgsonet al, 2002). Levels of active geminin are high during S, G 2 andmitosis, and this is important in preventing re-licensing of repli-cated D N A ( Q uinn et al, 2001; Mihaylov et al, 2002; Melixetianet al, 2004; Zhu et al, 2004; Li & Blow, 2005).

D irectly or indirectly, these inhibitory mechanisms ultimatelydepend on the increase in C D K activity that occurs at the end ofG 1 and persists unti l late mitosis (D iffley, 2004; N ishitani &Lygerou , 2004; Blow & D utta , 2005). The cell cycle can , there-fore , be thought of as consisting of two phases. The first phasecomprises late mitosis and early G 1 , when C D K levels are lowand licensing and cohesin loading occur. The second phase com-prises late G 1 , S, G 2 and early mitosis, when high C D K levelsdrive D N A replication and chromosome segregation (Fig 1).

In budding yeast, these different inhibitory pathways areredundant; to achieve re-replication of D N A , it is necessary to sta-bilize Cdc6 , remove inhibitory phosphorylation sites on ORC ,and prevent nuclear export of Mcm2–7 and Cdt1 (Nguyen et al,2001). In metazoans, downregulation of Cdt1 alone seems to bethe most important mechanism that prevents re-licensing of repli-cated D N A , as overexpression of Cdt1 or elimination of geminincan cause extensive re-replication ( Q uinn et al, 2001; Mihaylovet al, 2002; Vaziri et al, 2003; Zhong et al, 2003; Melixetian et al,2004; Zhu et al, 2004; Li & Blow, 2005). It is curious that meta-zoans, in which the consequences of genetic instability aregreater (as it might cause cancer), do not rely on the numerousmechanisms that are used by budding yeast. O ne possible expla-nation is that this might make it harder for genetically unstableclones to arise . If geminin were only one of several redundantmechanisms, its deletion might lead to clones that occasionallyre-replicate segments of D N A; however, being essential for theprevention of re-replication , geminin deletion is instead lethal .

S phaseMcm2–7 proteins are essential for replication of D N A during Sphase . They have sequence similarity with known D N A helicases(D N A-unwinding enzymes) and show D N A helicase activity in vitro (Blow & Dutta , 2005). Chromatin-immunoprecipitationexperiments in yeast have shown that during S phase , Mcm2–7proteins are displaced from replication origins and move ahead ofthe replication fork (Aparicio et al, 1997; Tanaka et al, 1997). IfMcm2–7 function is inhibited during S phase , the rate of D N Asynthesis rapidly falls, which indicates that Mcm2–7 proteins arerequired for the progression of replication forks (Labib et al, 2000;Pacek & Walter, 2004; Shechter et al, 2004). If Mcm2–7 proteinsform a replicative helicase that unwinds the template D N A aheadof the replication fork , this explains why they are displaced fromreplication origins when fork initiation occurs, thereby renderingthe origin unlicensed once it has been initiated .

O ne question left unanswered by this model is why so manyMcm2–7 hexamers are loaded onto the D N A (10–40 per origin).O ne possible explanation is that Mcm2–7 can act at a distancefrom the replication fork , pumping the D N A into the fork and

MM

M

M

Licensing

M

Cdc6 Cdt1

Cdt1Cdc6

ORC

ORC

ORC

Fig 2 | Components of the pre-replicative complex. The licensing of a singlereplication origin is shown.ORC is first recruited to chromatin, and thenrecruits Cdc6 and Cdt1. The ORC–Cdc6–Cdt1 complex licenses the origin byloading numerous Mcm2–7 complexes onto it.

(When) Each eukaryotic origin is licensed to initiate replication once and only once per cell cycle

J. Blow and T. Tanaka, EMBO Rep. 6:1028

MCM is a hexamericring that, similar to the sliding clamp, encircles DNA and can’t fall o!.

ORC (Origin Recognition Complex)binds to origins, marking each initiation site.

ORC then brings in additional proteins that load the MCM complex.

(When) Origin licensing is linked to the cell cycleLicensing is coupled to loading with cohesin, which holds newly-replicated chromosomes together until the daughter chromosomes are yanked apart during anaphase.

At G1/S, MCM loading proteins are is ubiquitylated and destroyed by the proteasome

At anaphase, cohesin is ubiquitylated and destroyed by the proteasome

review

©2005 EUROPEA N M OLEC ULAR BIOLO GY ORG A NIZATIO N EMB O reports V OL 6 | N O 11 | 2005

The chromosome cycleJ.J. Blow & T.U. Tanaka

1029

onto D N A . D uring S phase , the cohesin complex establishes aphysical l ink (cohesion) between sister chromatids. Cohesin isrequired for maintenance of this cohesion , and for ensuring thatsister chromatids are attached to microtubules extending fromopposite spindle poles (sister kinetochore bi-orientation) duringthe subsequent M phase (Tanaka et al, 2000; Sonoda et al, 2001).A t the metaphase-to-anaphase transition , after all chromosomesbi-orientate , a protease called separase is activated; this cleavesthe cohesin ring and allows the sister chromatids to be pulledapart by the mitotic spindle (Fig 1; U hlmann , 2003). The wholecycle of l icensing and cohesin loading is then able to start again .

Licensing and cohesin loadingCrystallography and electron microscopy of the archaealMcm2–7 homologue indicates that it forms a hexameric ringwith a positively-charged central channel that is able to encircledouble-stranded D N A (Fletcher et al, 2003; Pape et al, 2003). Theloading of Mcm2–7 onto D N A as an origin is l icensed mightinvolve the opening of the Mcm2–7 ring to allow it to encirclethe D N A . This would explain the high stability of Mcm2–7 com-plexes on D N A . A t least three other proteins—the origin recogni-tion complex ( O RC), Cdc6 and Cdt1—are required for Mcm2–7loading and the functional l icensing of D N A . Together, O RC ,

Cdc6 , Cdt1 and Mcm2–7 are components of the pre-replicativecomplex (pre-RC) of proteins that are found at replication originsduring G 1 .

The licensing reaction has been reconstituted on the chro-matin of Xenopus spermatozoa using purified O RC , Cdc6 , Cdt1and Mcm2–7 , in addition to a chromatin-remodelling proteinthat is required for the initial binding of O RC to D N A (G illespieet al, 2001). O RC binds to the D N A , and then recruits Cdc6 andCdt1 . O RC , Cdc6 and Cdt1 are required for the initial loading ofMcm2–7 onto D N A , but are not essential for the continued bind-ing of Mcm2–7 . This is consistent with O RC , Cdc6 and Cdt1 act-ing as a clamp loader that opens up the Mcm2–7 hexamer andcloses it again around the D N A (Fig 2). O RC , Cdc6 and Cdt1 areable to load several Mcm2–7 hexamers onto D N A , and eachreplication origin ( O RC-binding site) typically has 10–40Mcm2–7 hexamers associated with it by the end of G 1 (Blow &D utta , 2005).

The cohesin complex consists of four proteins: Smc1 , Smc3 ,Scc1 (also called Mcd1 and Rad21) and Scc3 (also called Psc3and SA1/2; Nasmyth & Haering, 2005). Each Smc molecule formsan intramolecular antiparallel coiled-coil , which brings theirglobular amino (N)- and carboxy (C)-terminal domains together(Anderson et al, 2002; Haering et al, 2002). Smc1 and Smc3dimerize through their central domains to form a V-shaped het-erodimer, which is closed by Scc1 and Scc3 to form a ring struc-ture . The cohesin complex is loaded onto chromosomes at theend of M phase in fission yeast and metazoan cells, and in late G1in budding yeast. This loading process requires a complex consist-ing of Scc2 (also called Mis4 and N ipped-B) and Scc4 (Ciosk et al,2000; Tomonaga et al, 2000). Cohesin loading also requires theATPase activity of Smc1 and Smc3 (Arumugam et al, 2003;Weitzer et al, 2003). Similar to licensing, this loading reactionmight involve opening up the cohesin ring and closing it againaround the D N A . In Xenopus egg extracts, the recruitment of Scc2to chromosomes depends on the presence of Mcm2–7 on thechromatin , which therefore tightly links these two processes(G illespie & H irano , 2004; Takahashi et al, 2004). However, inbudding yeast, cohesin loading takes place independently of pre-RC proteins and , in contrast to Mcm2–7 loading, can evenoccur after D N A replication (although , in this case , without con-tributing to sister chromatid cohesion; Uhlmann & Nasmyth1998; Haering et al, 2004). Cohesin association with chromo-somes is found at preferred sites, which include the regionsaround centromeres and sites between convergent transcriptionunits (Blat & Kleckner, 1999; Megee et al, 1999; Tanaka et al,1999; Tomonaga et al, 2000; Bernard et al, 2001; G lynn et al,2004; Lengronne et al, 2004). O ne possibility is that once loadedonto D N A , cohesin is redistributed to different places, perhaps bythe act of transcription itself.

Downregulation of licensing in late G1To prevent replicated D N A from becoming re-licensed and ,hence , re-replicated during a single S phase , it is crucial that theability to load new Mcm2–7 hexamers onto D N A ceases beforeentry into S phase . This can be achieved by downregulating theactivity of the O RC–Cdc6–Cdt1 loading machinery withoutaffecting Mcm2–7 that is already bound to D N A . D ifferent organ-isms regulate O RC , Cdc6 and Cdt1 activity in a range of ways(D iffley, 2004; N ishitani & Lygerou , 2004; Blow & D utta , 2005).

M

M

M

M

M

Metaphase Anaphase

M

M

M

M

MM

M

S

G1G2

Mcm2-7

Cohesin

Licensing and cohesin loading

Further licensing prevented

Fig 1 | Overview of the licensing and cohesion cycles. A small segment ofchromosomal DNA, encompassing three replication origins, is shown duringG1, S and G2.Mcm2–7 and cohesin are loaded during G1. During S phase, theMcm2–7 complex is displaced from DNA as it replicates, and cohesion isestablished. During anaphase, cohesin is cleaved, thereby allowing segregationof sister chromatids.J. Blow and T. Tanaka, EMBO Rep. 6:1028

MCM loadingproteinsdestroyed

Cell dividesDNA decondenses

DNA Synthesis

DNA condenses

Licensing oforigins: loadingwith MCM andcohesin

Decision to divide

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