Conserved Steps in Eukaryotic DNA Replication · CHAPTER 1 Conserved Steps in Eukaryotic DNA Replication XIN QUAN GE AND J. JULIAN BLOW Wellcome Trust Centre for Gene Regulation and

CHAPTER 1

Conserved Steps in EukaryoticDNA Replication

XIN QUAN GE AND J. JULIAN BLOW

Wellcome Trust Centre for Gene Regulation and Expression, University ofDundee, DD1 5EH, UK

1.1 Overview: the Biochemistry of DNA SynthesisThe genome of mammals comprises B6! 109 nucleotides arranged in extre-mely long linear polymers—the chromosomes. Accurate copying of thisamount of genetic information in a biologically relevant time frame (often a fewhours, though sometimes as little as a few minutes) requires highly accurateenzyme machines together with complex molecular coordination and feedback.The DNA template is a long polymer of four types of deoxyribonucleotide

arranged as a double-stranded anti-parallel helix (Figure 1.1A). The backboneof each strand consists of phosphodiester linkages between the 30 and 50 car-bons of deoxyribose. The 10 carbon of the deoxyribose is linked to one of fourdi!erent bases: the purines (adenine and guanine) or the pyrimidines (thymi-dine and cytosine). The two strands are held together by hydrogen bondsbetween complementary bases. The two-ringed heterocyclic purines alwaysbase pair with single ring pyrimidines, maintaining the linear axis of the helixand avoiding backbone distortion; specifically, guanine forms three hydrogen(H) bonds with cytosine and adenine makes two hydrogen bonds with thymi-dine (Figure 1.1B). Whilst each H bond is relatively weak, the huge number ofH bonds in an average mammalian chromosome (4109) ensures that the duplexis extremely stable. As noted by Watson and Crick in 1953,1 each of the two

Molecular Themes in DNA ReplicationEdited by Lynne S. Coxr Royal Society of Chemistry 2009Published by the Royal Society of Chemistry, www.rsc.org

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single strands of DNA contains all the information necessary to produce newsecond strands through complementary base pairing.During DNA replication, the two strands are opened up and a nascent

strand, complementary to the template strand, is synthesised by a complex ofmany proteins called the replication fork or replisome. Unwinding of the twostrands of DNA to expose bases during template-directed DNA synthesisrequires the input of chemical energy to break the hydrogen bonds. This energyis derived from hydrolysis of ATP by helicases, which act as DNA-dependentATPases that run ahead of the replication fork (Figure 1.2). In eukaryotes, themajor replicative helicase is almost certainly the hexameric Mcm2-7 complex2–5

(Figure 1.3, see also Chapter 3). At the same time, torsional stress (positivesupercoiling) is caused by unwinding the DNA and this is relieved by topoi-somerases (nicking-closing enzymes) (Figure 1.2). Then DNA is synthesised byenzyme-mediated polymerisation of deoxyribonucleotide triphosphates

Figure 1.1 The chemistry of DNA synthesis. (A) The duplex DNA template showingthe phosphodiester backbone, with the hydrophobic bases facing inwards,forming complementary base pairs. Reproduced from ‘An overview of thestructure of DNA’, created by Michael Strock 2006, released under theGNU Free Documentation Licence (GFDL).a (B) The four nucleotides ofDNA form hydrogen bonds with their complement (note opposite pola-rities of the two strands). Adenine pairs with thymidine via two H bonds,while guanine pairs with cytosine through three H bonds. (C) Phospho-diester bond formation through nucleophilic attack from the 30 OH of anewly incorporated nucleotide onto the a phosphate of an incomingnucleotide. The pyrophosphate released is rapidly hydrolysed by pyr-ophosphatase to inorganic phosphate, with a highly negative DG (B40kJmol"1). a

ahttp://en.wikipedia.org/wiki/File:DNA_Overview.png

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(dNTPs) complementary to the sequence of the exposed bases on the templatestrand (see Chapter 4). New daughter duplexes thus consist of one parentaltemplate strand base paired to a complementary daughter strand. This is semi-conservative replication and was first demonstrated experimentally by Mesel-son and Stahl,6 who showed that newly synthesised DNA is composed of onetemplate strand plus one nascent strand.During polymerisation, nucleophilic attack by the lone pair of electrons on

the 30 hydroxyl (OH) of deoxyribose onto the 50 phosphate of an incoming

Topoisomerase

Helicase

Figure 1.2 DNA duplex being unwound during DNA replication. Double-strandedDNA is being separated into two single strands by a helicase moving fromright to left. The topological interlinks between the two strands areremoved by a topoisomerase.

5'

3'5'

3'

DNA polymerase

Mcm2-7helicase

RNA primer

LEADING STRAND

LAGGING STRAND

5'3'

Okazakifragment

Figure 1.3 Leading and lagging strand replication. A replication fork is shown movingfrom right to left. The double-stranded template DNA is unwound by theMcm2-7 helicase. The leading strand is synthesised continuously in the 50–30 direction. The lagging strand is also synthesised in the 50–30 directionwith respect to the nascent strand, but since this is opposite to the overalldirection of fork movement, it is synthesised discontinuously in Okazakifragments. Each Okazaki fragment is started by a small RNA primerwhich is subsequently removed before the fragments are ligated together.

3Conserved Steps in Eukaryotic DNA Replication

dNTP results in the formation of a phosphodiester bond with the elimination ofpyrophosphate (Figure 1.1C). The subsequent, and very rapid, hydrolysis ofpyrophosphate to two inorganic phosphates releases energy, and it is this thatdrives the polymerisation reaction forwards. An important consequence of thisreaction is that DNA must always be synthesised in a 50 to 30 direction; allknown DNA polymerases act 50–30 with respect to the newly synthesised(nascent) DNA molecule. However, while the double-stranded DNA templateexists as an anti-parallel double helix, replication of both template strands iscoordinated at the replication fork, which moves in a net direction away fromthe start point (replication origin). To overcome this conflict of directionality,on only one strand (the ‘leading strand’) can DNA be polymerised in the samedirection as the fork is moving. On the other strand (the ‘lagging’ strand),nascent DNA is synthesised in short sections called Okazaki fragments, typi-cally B150 nucleotides long in eukaryotes (Figure 1.3). Okazaki fragments arestarted by short RNA primers which are subsequently removed before thefragments are joined together. Thus the fork can move away from the start sitewhile co-coordinating synthesis of both nascent strands and without contra-vening the energy requirements of 50–30 synthesis.In eukaryotes, the chromosomal DNA is located within the cell nucleus

where it is associated with proteins to form a DNA-protein complex calledchromatin (see Chapter 10). The basic building block of chromatin is thenucleosome core particle, which contains 147 base pairs of double-strandedDNA wrapped in 1.65 left-handed superhelical turns around the surface ofhistone octamer comprising two central H3–H4 dimers flanked on either sideby two H2A–H2B dimers (Figure 1.4). A variety of other proteins also bind toDNA and regulate its activity. For replication to occur, pre-existing nucleo-somes and other DNA-bound proteins that are located ahead of replicationforks need to be transiently disrupted. After fork passage, those proteins aredeposited back on parental as well as nascent DNA so that the chromatinstatus is reproduced in daughter strands7 (see also Chapter 10).

1.2 Where and When Does DNA ReplicationTake Place?

1.2.1 Cell Cycle Control

In eukaryotes, DNA replication takes place during a distinct phase of the cellcycle called S phase, during which time the entire genome is precisely duplicated(Figure 1.5). The replicated DNA molecules are segregated to the two daughtercells during a subsequent cell cycle phase called mitosis (see Chapter 9). S phaseand mitosis are separated by two ‘gap’ phases, G1 and G2. Progression througheach stage of the cell cycle is very tightly regulated by a complex interplay ofkinases (enzymes that phosphorylate proteins), phosphatases (enzymes thatremove phosphate groups from proteins) and proteases (which degrade pro-teins into shorter polypeptides or constituent amino acids).

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During S phase, pairs of replication forks are initiated bidirectionally fromchromosomal loci called replication origins. The large size of eukaryoticchromosomes (each of which can be tens or hundreds of megabases long) meansthat in order for them to be replicated in a reasonable period of time, a largenumber of replication origins are needed. Although the initiation of a pair repli-cation forks at a replication origin is a tightly controlled process, each fork willtypically then move along the DNA (‘elongate’) until it encounters a fork moving

H3-H4 dimer

H2A-H2B dimer

A.

B.

Figure 1.4 Structure of the nucleosome. (A) Cartoon of the nucleosome, showing 1.65turns of DNA wrapped around an octamer consisting of two H2A-H2Bdimers and two H3-H4 dimers. (B) Crystal structure of the nucleosomewith DNA: DNA in turquoise and brown, core histones H3 (blue),H4 (green), H2A (yellow) and H2B (red). Reprinted by permissionfromMacmillan Publishers Ltd: K. Luger, A. W. Mader, R. K. Richmond,D. F. Sargent and T. J. Richmond, Crystal structure of the nucleosomecore particle at 2.8A resolution, Nature, 1997, 389, 251–260, copyright(1997).84


in the opposite direction, at which stage both forks will disassemble (‘terminate’).When DNA is visualised during the S phase, replicated DNA can be observed as aseries of ‘bubbles’ with replication origins near their centres (arrowheads in Figure1.6). The stretch of DNA replicated by forks emanating from a single origin is

G1G2

S

meta-ana-

Mitosis

Origin Licensing(Mcm2-7 loading)

Inhibition ofLicensing

TimingDecision

Point

Replication of Different Chromosomal Domains

Figure 1.5 DNA replication and the cell cycle (schematic view of events). In the meta-phase of mitosis (M, meta-) condensed chromosomes (consisting of pairedchromatids) are aligned on the metaphase plate by the mitotic spindle.During anaphase (M, ana-) the two sister chromatids are pulled into twodaughter cells. During late mitosis and G1, replication origins are licensedby loading Mcm2-7 complexes. Origin licensing is inhibited at other cellcycle stages. During early G1, specific regions of chromosomal DNA takeup specific positions in the nucleus, with open chromatin (red) tending tobe positioned internally, and more condensed chromatin (blue) tending tobe positioned at the nuclear periphery and in larger internal structures. Atthis time (the timing decision point), these di!erent chromosomal regionsbecome programmed to replicate at di!erent stages of S phase (green).

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referred to as a replicon. Replicon sizes can vary significantly, both among dif-ferent organisms and among di!erent cell types in the same organism. Rapidlydividing cells typically have small replicon sizes (for example, cells in the earlyXenopus embryo has an average replicon size of B10kb, whilst mammaliansomatic cells typically have replicon sizes of 50–150kb8–10).

1.2.2 Origin Clusters and Replication Foci

In metazoans, adjacent origins (typically 2–5) are organised into clusters whichinitiate synchronously while di!erent origins clusters are activated at di!erentstages of S phase.11 One or more clusters of origins are organised into a discretereplication focal site, which has been estimated to comprise about 1 Mb of DNAand 6–12 replicons. Each focus is thought of as a factory for DNA replicationand contains a range of replication fork proteins (forming so-called repli-somes).12 It is possible that replisomes are anchored to a fibrous network withinthe nucleus (the ‘nuclear matrix’ or ‘nuclear sca!old’) through which multiplereplication forks are spooled; alternatively, the physical organisation of thechromosomal DNA into higher order chromatin structures could provide theframework on which replication foci are built.13–15 DNA replication is typicallycompleted in each focus within 30–120 minutes,16 and during this time, live cellimaging of the replication fork protein PCNAi (see Chapters 3 and 7) has shownthat replication foci do not merge, divide or have directional movement,17,18

thus arguing that replication foci are achieved by the coordinated assembly anddisassembly of replisomal proteins at sites that are more or less fixed.

1.2.3 The Replication Timing Programme

Eukaryotes replicate their genomic DNA according to a specific temporalprogramme, with di!erent clusters of origins firing at di!erent time during an Sphase that lasts from minutes in yeast to hours in metazoans. Several pieces ofevidence have suggested that chromatin context is a critical determinant oforigin initiation time. The replication timing programme is re-established in

0.1µM

Figure 1.6 Replication bubbles. Electron microscopic image of replication origins indeveloping Drosophila embryos. Replication bubbles are indicated by thearrowheads. Scale bar 0.1 mM. Reprinted from: G. Micheli, C. T. Baldari,M. T. Carri, G. Di Cello and M. Buongiorno-Nardelli, An electronmicroscope study of chromosomal DNA replication in di!erent eukar-yotic systems, Experimental Cell Research, 137, 127–140, copyright (1982),with permission from Elsevier.85

iProliferating cell nuclear antigen


each cell cycle shortly after mitosis.19 Transcriptionally active regions tend tohave open chromatin structure and replicate early, whereas gene-poor regionsand the more condensed heterochromatin replicate late.20,21 Transcriptionalsilencing can reprogramme an origin from initiating early to late, as well as bypromoting a more compact chromatin structure around the region.22

The timing decision point in early G1 (Figure 1.5) is the time when specificregions of the chromosome become programmed to replicate at specific stagesof S phase. This takes places coincidently with the repositioning of chromo-somes in the nucleus and the formation of immobile structures in the nucleusthat restrict chromosome mobility.19,23 It has been proposed that chromatinregulators might be concentrated into subnuclear compartments by a clusteringof related chromosomal domains, which may influence the timing of originfiring within a chromatin domain. For example in yeast, late replicatingorigins reside close to the nuclear periphery in G1, whereas early replicatingorigins are apparently randomly localised within the nucleus throughout thecell cycle.24

Many other factors could also contribute to determining the timing of originfiring. For example, in Saccharomyces cerevisiae, the timing of replication incertain origins is shown to be a!ected by the origin sequence.25 Precise levels ofcyclin-dependent kinase (CDK) activity present at various stages of S phase areimportant for executing the temporal programme. In budding yeast, two Sphase cyclins have di!erential roles in activation of early and late origins: Clb5activates both early and late origins, while Clb6 activates only early origins.26

The replication timing programme determines the di!erential firing time oflarge sequence blocks containing replication origin clusters, but why has the cellevolved such a sophisticated programme for DNA replication? The grouping ofreplication forks into factories that are activated at di!erent times might pro-vide an environment whereby newly replicated DNA could be assembled intospecific chromatin states, thus maintaining the epigenetic information that isimportant for regulation of other nuclear activities (such as transcription).10,27

It may also allow for tight regulation feedback, for example blocking firing oflate origins when replication from early origins is halted.

1.3 Origins of DNA Replication

The number of origins ranges from a few hundred in a yeast cell to tens ofthousands in a human cell. The extent to which conserved DNA sequenceelements determine origins di!ers significantly among eukaryotic species.Replication origins in the budding yeast Saccharomyces cerevisiae containhighly conserved sequence elements called A, B1, B2 and B3 boxes of theautonomously replicating sequence (ARS).28 These conserved DNA sequencesare required for binding of the initiator protein ORC (origin recognitioncomplex, see Chapter 2). However, not all DNA segments containing theconserved sequence elements are recognised by ORC in vivo. Other sequencesdistributed over 100 bp also contribute to replication origin function, possibly

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by providing binding sites for proteins that can enhance the recruitment ofORC to DNA or by providing DNA sequences that can be easily unwound.29

The origins in most other eukaryotes are much less stringent in terms ofsequence requirement. In the fission yeast, Schizosaccharomyces pombe, therequired origin sequences are distributed over large DNA segments (500–1000 bp) and are AT rich.30 It appears that it is the number of AT tracts in agiven segment of DNA that determines its probability of binding ORC andfunctioning as an origin of DNA replication.The nature of origins in metazoans is even less well defined than in yeasts and the

origins appear not to contain any consensus sequence. Replication origins occur atfrequent and nearly random intervals along metazoan chromosomal DNA, andonly a fraction of them are utilised in each cell cycle with a wide variation ofe"ciency. A typical pattern of origin initiation in metazoans is broad zones con-taining many relatively ine"cient origins, one or a few of which are selected sto-chastically and the rest are suppressed.31,32 However, at some origins, such aslamin B2 and b-globin origins, replication starts from tightly-defined sites.33,34

Several interacting components may influence the location and e"ciency ofinitiation in any given cell cycle, such as:

(1) DNA sequences. Sequences rich in AT could facilitate ORC binding orDNA unwinding.

(2) Local chromatin structure. It has been shown that the positions ofnucleosomes near origins are important for origin function.35 Whilsthistone acetylation has been shown to a!ect origin specification inXenopus and Drosophila,36,37 in mammalian cells, an ATP-dependentchromatin remodelling complex is required for e"cient replication ofheterochromatin.38

(3) Transcription. Transcription has been shown to interfere with originactivity and indeed, replication origins are almost never found withinactively transcribed DNA.10,20,39,40

(4) Protein–protein interactions. The presence of other proteins could helprecruit ORC and enhance origin e"ciency. For example, Abf1 and theMyb protein complex bind to origins and can a!ect the e"ciency oforigin utilisation in yeast and Drosophila.41,42

(5) Origin interference. It has been observed that in an initiation zone, firingof one replication origin appears to inhibit initiation at nearby origins,but is coordinated with neighbouring origins at more distant sites.43 Thismay suggest some sort of long range interaction between origins.

1.4 Licensing of DNA for ReplicationIt is essential for a cell to replicate its genome only once per cell cycle and this isregulated by the ability of cells to load the Mcm2-7 protein complex onto theorigins (see Chapters 2 and 3). Mcm2-7 form a clamp around DNA and providehelicase activity to separate the double helix ahead of replication forks2–5 (seeChapter 3). During late M and G1 phases of the cell cycle, Mcm2-7 are loaded


onto the DNA, which probably involves the clamping of the proteins aroundorigin DNA without activation of their helicase activity (Figure 1.7). This‘licenses’ the origin for use in the subsequent S phase. Mcm2-7 loading requiresthe recognition of the origin DNA by the origin recognition complex (ORC)(Figure 1.8). ORC in turn recruits proteins Cdc6 and Cdt1, which load Mcm2-7onto DNA by hydrolysing ATP44 (see Chapter 2). The complex of ORC, Cdc6,Cdt1 and Mcm2-7 at replication origins is termed the pre-replicative complex orpre-RC. It is not clear whether ORC, Cdc6 and Cdt1 open the Mcm2-7 ring andload it around DNA, or whether they facilitate the assembly of the Mcm2-7hexamer on DNA from di!erent Mcm subcomplexes present in the nucleoplasm.As a licensed origin initiates during S phase, the Mcm2-7 complex becomes

activated as helicase, possibly by binding other replication fork proteinsincluding the GINS complex.45,46 Since Mcm2-7 proteins travel with the repli-cation fork,47,48 this means that an origin becomes unlicensed after it initiates. Toprevent DNA being replicated a second time in a single cell cycle, it is thereforeimportant to prevent re-licensing of replicated origins during S and G2 phases ofthe cell cycle. The mechanisms for achieving this vary in di!erent eukaryotes.In yeasts, CDKs which are active from late G1 to mid-mitosis, prevent

licensing outside late M and G1 phase by ORC inactivation, Cdc6/Cdt1

M

M Free Mcm2-7

Mcm2-7On DNA

ActiveMcm2-7Helicases

ReplicationLicensing SystemInactive

ReplicationLicensing SystemActive

G1G2M

S

L

SM

M

M

M

M

MM

MM

CDK

g e m i n in

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dt1

+

M

M

R

Figure 1.7 The replication licensing cycle. The replication licensing system (RLS) isactivated in late mitosis, promoting the loading of Mcm2-7 double hex-amers onto DNA. During S phase, DNA-bound Mcm2-7 becomes acti-vated as helicases. When replication forks terminate, Mcm2-7 are releasedfrom DNA. In metazoans, the licensing system is shut down in S phaseand G2 by degradation of Cdt1 and activation of the Cdt1 inhibitor,geminin; in early mitosis, high CDK levels also inhibit licensing. In yeasts,licensing inhibition in S, G2 and early mitosis is directly mediated by highCDK levels.

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degradation and Mcm2-7 export.49 In metazoans, the main route by whichlicensing is prevented during S and G2 is the downregulation of Cdt1 activity.This is brought about both by degradation of Cdt1 protein and activation of aCdt1 inhibitory protein, geminin. Cdt1 is degraded at the end of G1 and early Sphase in a process dependent on SCF-class ubiquitin ligase and cul-4 ubiquitinligase.50–53 When geminin builds up during S, G2 and M phase, Cdt1 is sta-bilised by binding to geminin. As a result, licensing is inhibited and Cdt1 isprotected from degradation, so that when geminin is degraded in late mitosisand G1, Cdt1 is ready for licensing.49,54

ORC can load multiple copies of Mcm2-7 complexes onto DNA, and Mcm2-7 are inB20-fold excess over replication origins used in S phase.55–60 Cellssynthesise DNA at normal rates when the level of Mcm2-7 is reduced61,62 and,in Xenopus egg extracts, normal replication rates are maintained when Mcm2-7complex is reduced toBtwo per origin.59,60,63,64 This suggests that each of theloaded Mcm2-7 complexes could act at an origin to initiate DNA replicationand up to 90% of them remain dormant in a single S phase. It has recently beenshown that a biological role of the excess Mcm2-7 complexes loaded duringlicensing is to maintain genomic stability.65 When forks stall during DNAreplication, the dormant Mcm2-7 complexes can initiate and rescue DNAreplication between two stalled forks, allowing the intervening DNA to be

ORC

ORC

Cdc6 Cdt1

MM

ORC

Cdc6 dt1

M

A

B

C

Figure 1.8 Origin licensing. Cartoon showing steps in the licensing of a replicationorigin. (A) ORC association with DNA at the replication origin. (B) ORCrecruits Cdc6 and Cdt1. (C) ORC-Cdc6-Cdt1 allows the loading of mul-tiple Mcm2-7 complexes onto the DNA.


replicated. If a replication fork encounters an unfired (dormant) origin, theMcm2-7 must be removed from it to prevent re-replication from occurring.

1.5 Initiation of DNA ReplicationMcm2-7 is loaded in an inactive form at replication origins during G1 (Figure1.9A), and then activated to initiate DNA replication during S phase. Acti-vation of Mcm2-7 requires both S phase CDK (cyclin E/A-Cdk2) and Dbf4-dependent kinase (DDK) activity (Figure 1.9B). DDK and CDK are expressedat relatively constant levels during the cell cycle, but the expression of reg-ulatory subunits (Dbf4 and cyclin respectively) is increased in S phase. One ofthe known substrates of Cdc7-Dbf4 is the Mcm2-7 complex, the phosphor-ylation of which is thought to change its interaction with other replication forkproteins.66 Recently, it has been shown in budding yeast that S phase CDKsphosphorylate two replication proteins, Sld2 and Sld346,67 (Figure 1.9B). Bycontrast, the critical S phase substrates of CDK-cyclin activity in metazoanshave not yet been identified. Phosphorylation of yeast Sld2 induces its inter-action with Dbp11, and facilitates its association with the GINS complex (Sld5,Psf1, Psf2, Psf3) and Dbp11 (Figure 1.9C). At the same time, Sld3 is phos-phorylated by CDK and recruited to DNA by binding to Cdc45, wherephospho-Sld3 recruits Dbp11 and Sld2. Current evidence suggests that thebinding of Cdc45 and GINS to Mcm2-7 activates the helicase activity ofMcm2-7.45,46 Consequently GINS and Cdc45 remain associated with Mcm2-7and travel with the active replication forks (see Chapter 3).

1.6 Elongation of Replication Forks

Mcm2-7 and associated complex unwind the DNA in a bidirectional manneraway from origins, and the single-stranded DNA (ssDNA) becomes coated witha binding protein called Replication Protein A (RPA). Via interactions with thehelicase and RPA, DNA polymerase a (pola) is loaded onto the template(Figure 1.9D). A subunit of the pola holoenzyme provides primase activity andsynthesises short RNA primers (8–12 nucleotides long), which are then extendedby the DNA polymerase activity of pola to synthesise a short initiator DNA(iDNA) of about 30 bases. Because pola does not have a proofreading exonu-clease activity, the iDNA synthesised only serves as a DNA primer for moreextensive DNA synthesis by DNA polymerases with proofreading activity afterpolymerase switching (Chapter 4). The primer-template DNA structure isrecognised and bound by a clamp-loading heteropentameric protein complex,replication factor C (RFC). This promotes structural changes in RFC, whichuses the energy from ATP hydrolysis to open the ring of the trimeric slidingclamp PCNA (Chapter 2), and clamp it around the DNA, while at the same timepola is displaced. PCNA acts as a processivity factor for the elongation DNApolymerases pold and pole by forming a ring that tethers them to the templateDNA (Chapters 3, 6 and 7). Current data suggest that pole is on the leading

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Sld3 Sld2PP Dpb11

Cdc45 pol!G

I NSP P

Ppol!G

I NS

GI NS

p o l!

p o l"p o l#

pol#pol"C d c4 5

Cdc45

5'3'

LicensedOrigin

Mcm2-7

5'3'

5'3'

5'3' 5'

3'

5'3'

5'3'

5'3'

P

Cdc7

Sld3 Sld2PP Dpb11

Sld3P

Sld2P

CDK

A

B

C

D

Figure 1.9 Initiation of replication forks. A model for events occurring during S phaseas an origin initiates DNA replication. (A) A licensed origin, loaded withtwoMcm2-7 hexamers. (B), Cdc7 phosphorylates members of the Mcm2-7complex, whilst CDKs phosphorylate Sld2 and Sld3. Phosphorylated Sld2and Sld3 associate with Dpb11. (C) The Sld2-Sld3-Dpb11 complexassociates with phosphorylated Mcm2-7, possibly via interactions betweenSld3 and Cdc45 and between Sld2 and GINS/pole. (D) Mcm2-7 helicaseactivity unwinds the origin DNA, allowing pola to initiate synthesis of thetwo nascent strands. These nascent strands are elongated by pole to formthe leading strand of the fork. Behind this, pola and pold act together tosynthesise Okazaki fragments on the lagging strand.


strand and pold is on the lagging strand.68,69 When the lagging strand poly-merase encounters the 50 end of the adjacent Okazaki fragment, the 50 end isdisplaced to form a 50 flap, which is degraded by endonuclease FEN1 (Chapter5). Then the two Okazaki fragments are ligated by DNA ligase and the DNApolymerase is recycled to a newly loaded clamp on the lagging strand.70

Recent work in budding yeast indicates that Mcm2-7 associate with anumber of proteins at forks to form the ‘replisome progression complex’ (RPC)during elongation,71 perhaps to ensure that fork progression is coordinatedwith DNA synthesis and other processes. In addition to Cdc45 and GINS, theRPC also contains Ctf4 which is important for establishing cohesion betweensister chromatids, Tof1-Csm3, which mediate pausing of forks at DNA repli-cation fork barriers, the checkpoint mediator Mrc1, the histone chaperoneFACT, topoisomerase 1 and Mcm10.72 In addition, PCNA acts as a landingpad for other proteins during replication such as the CDK inhibitor p21,cytosine methyltransferase, the chromatin assembly factor CAF-1, DNA ligase,FEN1 and other proteins involved in DNA repair73–75 (see Chapter 3).

1.7 Termination of DNA ReplicationReplication forks terminate when they encounter another replication forkcoming from the opposite direction. In most cases, this occurs without the needfor any special DNA sequences. In some cases, though, replication fork barriersat specific DNA sequences slow replication forks so that these sites are likely tobecome sites of termination. One such example is in the heavily transcribedribosomal DNA genes, where the replication fork barrier is positioned toinhibit replication forks from moving through the gene in the opposite directionfrom transcription.76

The exact mechanism of how replication machinery is displaced from theDNA during termination is poorly understood. At termination, the replicationforks must be disassembled. Most of the proteins released from terminatedreplication forks can be recycled to newly initiating forks. The Mcm2-7 pro-teins, however, are a special case. They are released from DNA at termination,but are not reloaded onto DNA until the next mitosis in order to prevent DNAfrom being replicated more than once in a single cell cycle (Figures 1.5 and 1.7).Similarly, if an active fork encounters inactive Mcm2-7 bound to a dormantreplication origin, the inactive Mcm2-7 will be displaced from DNA.

1.8 Replication of ChromatinThe histones around which the DNA is wrapped (Figure 1.4) have to be dis-placed from the chromatin as the DNA replication forks pass, and the newlysynthesised DNA has to be reassembled into chromatin (see Chapter 10).Nucleosome disruption is likely to be facilitated by ATP-dependent chromatinremodelling enzymes, such as WSTF which is targeted to replicating DNAthrough direct interaction with PCNA and in turn recruits ISWI-type

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nucleosome-remodelling factor SNF2.77 At the same time, histone chaperonesfacilitate the disruption of parental nucleosomes by acting as histone acceptorsand hence aid the transfer of the histones onto the nascent strand.78 FACT iscomplexed with Mcm proteins during fork movement, and facilitates nucleo-some disruption and re-deposition of H2A-H2B.79,80 CAF-1 associated withPCNA is aided by Asf1 to deposit H3-H4 onto replicating DNA.81,82

Chromatin also contains epigenetic information in addition to that from theDNA sequence which a!ects, amongst other things, the level of gene expres-sion. This information is encoded by covalent modifications to histones (suchas acetylation and methylation) as well as methylation of cytosine bases. WhenDNA is replicated, the epigenetic information must be copied too. This isachieved by association of a large number of chromatin-modulating enzymeswith PCNA during replication such as DNA methyltransferase I, CAF-1 andhistone deacetylase (Chapters 3 and 10). These enzymes either themselves havecatalytic activity or can recruit other enzymes implicated in chromatinmodification.

1.9 Chromatid Cohesion and Segregation

After replication, it is essential that the sister chromatids are identified and eachof them is sent to a di!erent daughter cell. To achieve this, replicated chro-mosomes remain physically attached to each other by cohesion until anaphase,when they are separated by microtubule pulling force. Sister chromatid cohe-sion is established by cohesin, a complex consisting of at least four proteins(Smc1, Smc3, Scc1, Scc3) that form a ring structure loaded at discrete sitesalong the entire length of the chromosome in G1 phase. During S phase, thecohesin complex establishes a physical link (cohesion) between replicated sisterchromatids by several factors, including Eco1, Ctf4 and Ctf18. Several modelshave been proposed to explain how cohesin contributes structurally to sisterchromatid cohesion, one of which is that the cohesin ring establishes cohesionby embracing both sister chromatids (see Chapter 9).At the metaphase-to-anaphase transition, the separation of sister chromatids

is triggered by the removal of cohesin from chromosomes. This is achieved byactivation of a protease called separase, which cleaves the cohesin ring.Separase is inhibited by protein securin and, at the metaphase-to-anaphasetransition, securin is degraded after APC/C dependent ubiquitination.83 Thus acomplex interplay of cell cycle regulatory factors establishes cohesion con-comitant with synthesis of sister chromatids and ensures separation only at themetaphase-anaphase transition (Chapter 9).

Acknowledgements

The authors are supported by Cancer Research UK grants C303/A4416 (XQG)and C303/A7399 (JJB).


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Conserved Steps in Eukaryotic DNA Replication · CHAPTER 1 Conserved Steps in Eukaryotic DNA Replication XIN QUAN GE AND J. JULIAN BLOW Wellcome Trust Centre for Gene Regulation and