Regulation of S Phase

This complex includes the Origin Recognition Complex ORC, which serves to recognize origins, the putative helicase MCM2-7, and other factors important for com- plex assembly.. Otherfacto

P Kaldis: Cell Cycle Regulation

DOI 10.1007/b137221/Published online: 6 July 2005

© Springer-Verlag Berlin Heidelberg 2005

Regulation of S Phase

Jamie K Teer1,2· Anindya Dutta1,2(u)

1 Biological and Biomedical Sciences Program, Harvard Medical School,

Boston, MA 02115, USA

ad8q@virginia.edu

2 Dept Of Biochemistry, University of Virginia, Charlottesville, VA 22908, USA

ad8q@virginia.edu

Abstract Regulation of DNA replication is critical for accurate and timely dissemination

of genomic material to daughter cells The cell uses a variety of mechanisms to control this aspect of the cell cycle There are various determinants of origin identification, as well as a large number of proteins required to load replication complexes at these defined genomic regions A pre-Replication Complex (pre-RC) associates with origins in the G1 phase This complex includes the Origin Recognition Complex (ORC), which serves to recognize origins, the putative helicase MCM2-7, and other factors important for com- plex assembly Following pre-RC loading, a pre-Initiation Complex (pre-IC) builds upon the helicase with factors required for eventual loading of replicative polymerases The chromatin association of these two complexes is temporally distinct, with pre-RC being inhibited, and pre-IC being activated by cyclin-dependent kinases (Cdks) This regulation

is the basis for replication licensing, which allows replication to occur at a specific time once, and only once, per cell cycle By preventing extra rounds of replication within a cell cycle, or by ensuring the cell cycle cannot progress until the environmental and intracel- lular conditions are most optimal, cells are able to carry out a successful replication cycle with minimal mutations.

to ensure that the proper environment exists to carry out replication Thesemechanisms can be divided into proper identification of appropriate origins

of replication, and subsequent loading of the replication machinery itself Wewill focus our discussion on the initiation of replication in eukaryotes, andhow it can be used by the cells to control S-phase progression

Origins of Replication

2.1

Genome Replicator Sequences

Theoretically, replication would be carried out most efficiently by startingfrom many different evenly spaced sites Such a model might assume, how-ever, that specific, conserved origins of replication exist Studies on origins in

various systems support the early theory of a replicator sequence that marks the origin of replication, and an initiator protein that binds this sequence and

recruits downstream factors required for replication (Jacob et al., 1963) The

earliest eukaryotic replicator was found in Saccharomyces cerevisiae, and was

named ARS for autonomously replicating sequence (Stinchcomb et al., 1979;Struhl et al., 1979) Study of the ARS1 locus in budding yeast revealed con-served sequence blocks that were essential for replication, including an 11 bpconsensus seqeunce (A element) and several other B elements (Marahrensand Stillman, 1992) These replicator sequences were used later to identify theputative initiator proteins: the Origin Recognition Complex [ORC] (see be-

low) In Schizosaccharomyces pombe, two 30-55 base pair elements essential

for replication were discovered in origin ars3002, and similar sequences werefound in other ars regions (Dubey et al., 1996) Although the yeasts seem tohave high sequence conservation from one replicator to the next, determiningsuch consensus replicators in higher eukaryotes has been more difficult

Studies in Xenopus laevis egg extracts have not identified a consensus

replicator sequence On the contrary, early results indicate the lack of quence specificity in replicating regions (Hyrien and Mechali, 1992; Hyrienand Mechali, 1993; Mahbubani et al., 1992) Recent studies show that whilethe ORC proteins may prefer AT rich DNA stretches, they show no preferencebetween defined origin sequences and control sequences in vitro, even withvarying ORC concentration (Vashee et al., 2003) Such random origin selec-tion may, however, be a function of the early embryogensis system When ori-gin selection in the rDNA locus was studied at different times in development,increasing origin specificity was seen as development progressed (Hyrien

se-et al., 1995) In early stages, origin selection was random, but when rDNAgene expression began in late blastula and early gastrula stages, initiationfrequency decreased in the transcribed regions This effectively limited ini-

tiation to the intergenic regions Similar results were observed in Drosophila

embryos (Sasaki et al., 1999) Interestingly, when intact mammalian nuclei

were added to Xenopus extracts, they initiated replication at specific sites.

Disrupting the nuclei before incubation ablated this specificity (Gilbert et al.,1995) Additionally, intact mammalian nuclei isolated before a certain time inthe G1 phase also failed to initiate specifically (Wu and Gilbert, 1996) Taken

together, these results indicate that metazoans do seem to initiate replication

at specific sites, but this specificity may be determined not by sequence, but

by other influences from local chromatin and nuclear environments

The picture is also complicated in mammalian systems Early work inchinese hamster ovary cells revealed a replication origin in the dihydrofo-late reductase (DHFR) locus (Heintz and Hamlin, 1982; Heintz et al., 1983).Although this origin firing was originally thought to be highly sequence spe-cific, later two dimensional gel electrophoresis showed that origins fire in

a broad zone (55 kb) throughout the intergenic region [but not in the DHFRgene itself] (Dijkwel and Hamlin, 1995; Vaughn et al., 1990) These observa-tions argue against a defined sequence specificity A different study, however,used nascent strand abundance assays on the same DHFR region to demon-strate only two to three major initiation sites, again raising the possibility ofsequence specificity (Burhans et al., 1990; Kobayashi et al., 1998) Recently,

a study using early labeled fragment hybridization (ELFH) showed that theearliest nascent strands could hybridize to many clones from different areasalong the intergenic region, suggesting that replication can be initiated frommany different sites (Dijkwel et al., 2002) Additionally, a deletion mappingexperiment showed that replication could initiate from the intergenic region

in the absence of the major sites, and even in the absence of 90% of the region(Mesner et al., 2003) These studies indicate the regions of potential origin fir-ing in higher eukaryotes may not be determined simply by sequence, but byother factors

Like Chinese hamster cells, few origins of replication are known in humancells One of the earlier defined origins of replication is at theβ-globin lo-

cus A bidirectional origin was found to exist in the 2 kb region between the

δ and β globin genes, and deleting this region abrogated the bidirectional

ac-tivity (Kitsberg et al., 1993) This result suggested a sequence element may bepresent in this region to direct origin firing Furthermore, an ectopically in-sertedβ-globin locus promoted initiation at the ectopic site (Aladjem et al.,

1998) Deletion mapping of theβ-globin locus showed several sequences

crit-ical for replication initiation at the locus, in both ectopic and native locations(Aladjem et al., 1998; Wang et al., 2004) Similar results have been observed

at the lamin B2 locus [1.2 kb] (Paixao et al., 2004), the hamster DHFR cus [5.8 kb] (Altman and Fanning, 2004), and the c-myc locus [2.4 kb] (Liu

lo-et al., 2003): ectopically inserted sequences can confer origin activity, anddeletion of specific elements eliminates such activity Unfortunately, the se-quence elements do not seem to be identical, and no consensus sequenceshave emerged There does seem to be an important role of AT rich sequences,

as these are often found in critical deleted regions Supporting this idea, anessential AT rich element in the lamin B2 locus can substitute for the AT-rich element in hamster ori-β locus (Altman and Fanning, 2004) One should

note that experiments showing sequence specificity generally measure originfiring by PCR of nascent strands, while studies supporting sequence inde-

pendent origin firing use ELFH and two dimensional gel electrophoresis Thepossibility exists that different methodologies may have different effects onthe results.

In addition to sequence effects, many studies have implicated tion in selection of replication origins In the DHFR locus, transcription ofDHFR itself is required for origin firing activity, and yet origins do not fire inthe gene (Kalejta et al., 1998; Saha et al., 2004) In yeast, evidence exists fortranscriptional correlation with replication (Muller et al., 2000), but this may

transcrip-be limited to few specific sites, as a genomic microarray study failed to see

a good correlation (Raghuraman et al., 2001) Many studies have shown a linkbetween early origin firing and transcriptional activity by looking at replica-tion of developmentally regulated genes, as well as genes from asymmetricallyactive alleles [reviewed in (Goren and Cedar, 2003)] In the latter case, theactive alleles are replicated much earlier than the silenced alleles Addition-ally, replication studies using human (Jeon et al., 2005; Woodfine et al., 2004)

and Drosophila (Macalpine et al., 2004; Schubeler et al., 2002) genome tiling

microarrays show a positive correlation between early origin activity, gene

density, and transcriptional activity Recent results in Drosophila indicate that

histone hyperacetylation is important for ORC recruitment, although inducedhyperacetylation did not affect transcription (Aggarwal and Calvi, 2004).These studies indicate that, in higher eukaryotes, an environment generated

by transcription allows for efficient origin firing One might imagine that theopen chromatin environment for transcription would also benefit replication,linking the two different activities

Although controversy exists as to whether higher eukaryotic origins aresequence dependent or not, the complexity of these organisms may allow a re-ality that lies somewhere in the middle In budding yeast, sequence specificity

in the form of well established consensus replicators seems to be the primarydeterminant of origin locations However, as organism complexity increases,

so does the complexity of origin determination In metazoans, a consensusorigin sequence has not yet been identified Many reports show that certainsequence elements are important for firing, but that these elements for themost part do not share common primary sequence, or even overall features,aside from AT rich sequence preference Initiator proteins show no prefer-ence for sequence, but may show some preference instead for structure [to

be discussed later] (Remus et al., 2004), indicating that these essential ents exist to provide a favorable environment for initiator loading Otherfactors may also affect chromatin structure, especially during transcription.The positive correlation between origin firing and transcriptional activity inhigher eukaryotes suggests that the more open chromatin structure not onlyallows efficient transcription, but efficient replication initiation as well It isalso plausible that different origins may have differential influences on theiractivity Nearby transcriptional activity may be important for one origin,whereas critical sequence elements are important for another In summary,

elem-origin selection in eukaryotes is defined by the proper environment for tiator binding, whether defined solely by DNA sequence elements, structuralelements, chromatin structure itself, or a combination of effects Thus, the

ini-early theory of a replicator still holds true today The increasing complexity

of higher eukaryotes simply means that the defining elements of a replicator

are themselves more multifaceted

3

Pre-Replication Complex

3.1

ORC

The identification of replicator sequences in S cerevisiae opened the field of

DNA replication in eukaryotes One of the first critical discoveries stemming

from this work was the identification of the proposed initiator proteins The

consensus A element of the ARS sequence was used to identify a six subunitcomplex termed ORC, or Origin Recognition Complex (Bell and Stillman,1992) Mutations in the A element that prevent ORC binding also preventreplication from the mutated ARS, (Bell and Stillman, 1992; Rowley et al.,

1995) supporting the idea that budding yeast ORC is the protein initiator responsible for recognizing specific replicator sequences ORC is highly con- served, with homologues identified in A thaliana, S pombe, D melanogaster,

X laevis, M musculus, H sapiens, and others (Carpenter et al., 1996; Dhar

and Dutta, 2000; Gavin et al., 1995; Gossen et al., 1995; Leatherwood et al.,1996; Masuda et al., 2004; Muzi-Falconi and Kelly, 1995; Pinto et al., 1999;Quintana et al., 1997; Quintana et al., 1998; Tugal et al., 1998) The ORC

subunits have been shown to form a functional complex in D melanogaster (Chesnokov et al., 1999), S pombe (Moon et al., 1999), X laevis (Gillespie

et al., 2001), and H sapiens (Dhar et al., 2001a; Vashee et al., 2001).

As a replicative initiator, ORC should be able to recognize the replicator

sequences ORC has been shown by many to bind DNA, and this binding isdependent on ATP and the ATP binding functions of ORC (Bell and Still-man, 1992; Chesnokov et al., 2001; Gillespie et al., 2001; Seki and Diffley,

2000) Specific replicator sequence association has been observed in S visiae and S pombe Work in the latter organism has revealed that Orc4

cere-dictates the specificity via a newly defined AT hook region (Kong and Pamphilis, 2001; Lee et al., 2001) ORC from higher eukaryotes, however, doesnot seem to have the same sequence specificity Drosophila ORC showed lit-tle preference for chorion gene sequences compared to controls, but showed

De-a much better preference for negDe-ative supercoiled DNA compDe-ared to relDe-axed

or linear, suggesting secondary structure is more important than sequence for

initiator/replicator interactions in metazoans (Remus et al., 2004) Similarly,

human ORC shows no preference for known origins compared to randomDNA sequences, but does show a slight preference for AT rich DNA (Vashee

et al., 2003) While ORC may be responsible for DNA binding, the ism of such binding becomes unclear with increasing organism complexity,perhaps due to the increasing complexity of factors affecting origin selection.Although the intricacies of the ORC-DNA interaction are not fully un-derstood, the general role of ORC in replication is now accepted Studies in

mechan-Xenopus egg extracts have demonstrated that ORC is required to load Cdt1

and Cdc6, themselves factors required for replication initiation [discussedfurther below] (Coleman et al., 1996; Maiorano et al., 2000) This dependence

of replication factor recruitment on ORC helps to explain the lethality of allORC subunit deletions in yeast As the foundation of replication initiationcomplexes, the role of ORC seems to be critical for downstream functions

As ORC is a key player in defining and recruiting a replication complex

to an origin, it is an important potential target for controlling replication

In yeasts, the ORC remains bound to chromatin throughout the cell cycle

However, in S cerevisiae, Orc2 and Orc6 are phosphorylated by S-phase

cyclin/Cdk1 during the G1/S transition This ORC phosphorylation was

found to be part of a mechanism to limit origin firing activity to only onceper cell cycle; when Orc2 and Orc6 phosphorylation site mutants were intro-duced with constitutively active Cdc6 and MCM proteins (see Sects 3.3 and3.4, respectively), rereplication was observed (Nguyen et al., 2001; Wilmes

et al., 2004) Similarly, S pombe Orc2 is phosphorylated, which may be due

to its similar interaction with Cdk1/cyclin B in the G2 phase This interaction

serves to prevent rereplication without an intervening mitosis, again ing only one replication event per cell cycle takes place (Wuarin et al., 2002).Phosphorylation is well studied as a molecular switch to regulate protein ac-tivity through a variety of mechanisms and ORC phosphorylation gives thecells a reversible way to prevent replication firing

ensur-In higher eukaryotes, phosphorylation of ORC subunits is also observed

In Xenopus systems, phosphorylation of ORC by cyclin A dependent kinase

activity disrupts ORC chromatin association (Findeisen et al., 1999) larly, mammalian Orc1 is phosphorylated In Chinese hamster ovary cells,Orc1 interacts with cyclin A/Cdk1, which leads to the phosphorylation of

Simi-Orc1 Inhibiting this phosphorylation with drugs allows Orc1 to rebind matin, indicating that phosphorylation is important for chromatin release inmitosis (Li et al., 2004) In human cells, Orc1 is also phosphorylated in vivo(our unpublished results) and in vitro by cyclin A/Cdk2 (Mendez et al., 2002).

chro-This phosphorylation seems to be required for Skp2 mediated ubiquitination

of Orc1 (Mendez et al., 2002) Similarly, hamster Orc1 is also ubiquitinated.However, the nature and effect of these ubiquitination events is different Inhamster cells, Orc1 seems to be mono- and di-ubiquitinated, which causes itsrelease from chromatin in S-phase until M-G1 (Li and DePamphilis, 2002)

In humans, several studies show that Orc1 is polyubiquitinated and then graded by the proteasome during S-phase, (Fujita et al., 2002; Mendez et al.,2002; Tatsumi et al., 2003) although this observation may result from proteol-ysis after lysis (Ritzi et al., 2003) Although not degraded during the cell cycle,hamster Orc1 is increasingly sensitive to proteasomal degradation when ar-

de-tificially released to the cytoplasm (Li and DePamphilis, 2002) In Drosophila

embryos, Orc1 is degraded in M and early G1 phases by the APC/fzr complex

(Araki et al., 2003) Despite the apparent contradictions, which may simplyresult from differences between organisms or even cell types, Orc1 binding tochromatin can be regulated in higher eukaryotes This regulation allows thecells to control replication initiation at the basic level of the ORC, ensuringinappropriate replication initiation has little, if any, chance of success

3.2

Cdt1

Cdt1 was first identified as a Cdc10 regulated gene in S pombe This gene was

found to be cell cycle regulated, and important for replication (Hofmann andBeach, 1994) It associates with Cdc6 and is required for loading of the MCMcomplex in several model systems (Maiorano et al., 2000; Nishitani et al.,2000; Tanaka and Diffley, 2002) Its own chromatin loading is dependent onORC (Maiorano et al., 2000), supporting the idea that ORC serves as a foun-dation that recruits downstream factors for replication Cdt1 protein itself isregulated during the cell cycle, not by transcription, but by proteasome de-pendent degradation in S-phase (Hofmann and Beach, 1994; Nishitani et al.,

2001) A study in C elegans implicated the ubiquitin ligase Cul-4 in Cdt1

degradation; when Cul-4 is absent, Cdt1 is stabilized in S-phase, and sive re-replication is observed (Zhong et al., 2003) In humans, the SCFSkp2complex is implicated in the destruction of Cdt1, and is dependent uponphosphorylation by cyclin-dependent kinases [Cdks] (Li et al., 2003b; Sugi-moto et al., 2004), although mutations in Cdt1 that disrupt association withSkp2 still permit degradation of Cdt1 in S-phase (Takeda et al., 2005) Re-

mas-cent work in Xenopus egg extracts indicates that the degradation of Cdt1

after replication initiation, together with its inhibition by the protein nin (discussed later), limit replication to a single round per cell cycle (Ariasand Walter, 2004; Li and Blow, 2004) As Cdt1 is required for pre-RC loading,its inhibition by either geminin interaction or degradation will help preventfurther pre-RC formation, and thus, further replication initiation Consistentwith this, overexpression of Cdt1 alone leads to extensive re-replication inhuman cells (Vaziri et al., 2003)

gemi-In addition to its function as a replication licensing factor, Cdt1 has cently been implicated in preventing replication initiation after DNA damage

re-in human cells Cdt1 levels were found to be profoundly decreased after UVirradiation, and an E3 ligase, Cul4A-Roc1-Ddb1, was responsible for signaling

this degradation via the proteasome (Higa et al., 2003; Hu et al., 2004) terestingly, a separate study implicated the SCFSkp2complex in the radiationinduced degradation of Cdt1 (Kondo et al., 2004) It remains to be resolvedwhich ubiquitin ligase is primarily responsible for both the cell cycle depen-dent modifications and the DNA damage induced modifications.

In-3.3

Cdc6

Cdc6 was originally identified in S cerevisiae as a protein essential for cell

cycle progression (Hartwell et al., 1974), and was thereafter shown to have anearly DNA synthesis defect (Hartwell, 1976) Cdc6 interacts with ORC, form-ing a complex with an extended nuclease protected DNA footprint (Cocker

et al., 1996; Liang et al., 1995) Furthermore, Cdc6 expression is required forMCM loading in budding yeast Interestingly, phosphorylation of Cdc6 by B-type cyclin/Cdk complexes prevented the loading of Cdc6, illustrating a pow-

erful way for the cells to regulate pre-RC formation (Donovan et al., 1997;Tanaka et al., 1997) By phosphorylating Cdc6 in S and G2/M, its activity was

limited, preventing inappropriate origin firing in mitosis ScCdc6 was alsofound to be marked for degradation at the G1/S transition by Clb/Cdc28 and

the Cdc4/Cdc34/Cdc53 ubiquitination machinery, adding a further layer of

regulation (Drury et al., 1997; Elsasser et al., 1999) A similar gene, Cdc18, was

identified in S pombe, and is also required for S-phase Indeed, its pression in S pombe resulted in severe rereplication Additionally, its protein

overex-levels cycle, with maximum overex-levels present during the G1/S transition

(Muzi-Falconi et al., 1996; Nishitani and Nurse, 1995) Cdc18 is phosphorylated uponentry into S-phase, causing its rapid degradation (Jallepalli et al., 1997) Notonly is Cdc18 required for MCM binding, but it seems to promote this binding

in anaphase, supporting the idea that pre-RCs are formed in mitosis (Kearsey

et al., 2000) Cdc18 and Cdc6 were later shown to be homologues

Cdc6 is found in higher eukaryotes as well Homologues have been

identi-fied in Xenopus, humans and others (Coleman et al., 1996; Saha et al., 1998; Williams et al., 1997) Using the Xenopus egg extract system, it was found

that Cdc6 binding to chromatin is dependent on ORC, and is required forMCM2-7 loading, thus implicating a sequential assembly of pre-RC compo-nents (Coleman et al., 1996) Similar results were observed in a human cellfree extract (Stoeber et al., 1998) Human Cdc6 is partially cell cycle regulated;

it is under the control of the E2F transcription factor, which is responsible forpromoting expression of numerous genes required for proliferation (Ohtani

et al., 1998; Yan et al., 1998) However, unlike yeasts, human Cdc6 may not

be degraded at the G1/S transition, but has been found to be exported from

the nucleus in a phosphorylation dependent manner (Delmolino et al., 2001;Fujita et al., 1999; Jiang et al., 1999; Saha et al., 1998) This phosphoryla-tion is controlled by cyclin A/Cdk2, which allows for export of Cdc6 soon

after replication has initiated (Petersen et al., 1999) Some evidence exists thathuman Cdc6 is degraded in Sphase, just as in yeast, a point that will needresolution (Coverley et al., 2000; Mendez and Stillman, 2000) Recently it wasalso reported that although exogenous Cdc6 is exported from the nucleus inS-phase, endogenous Cdc6 is not (Alexandrow and Hamlin, 2004) One fur-ther study demonstrates that although Cdc6 is released from chromatin andthen degraded, it is constantly being resynthesized and immediately bindschromatin, replacing molecules which were displaced (Biermann et al., 2002).This observation may reconcile earlier observed differences, allowing Cdc6 tobind chromatin in S-phase in a tenuous manner so that rapid regulation can

be achieved when needed

Cdc6 is a AAA+ ATPase as is its homolog Orc1 (Neuwald et al., 1999), and

is therefore also regulated in cis The recently solved structure of an archaeal

Cdc6 ortholog confirms the presence of a AAA+ ATPase domain, ing Walker A and B motifs, and well as several sensor regions thought todetect nucleotide binding status (Liu et al., 2000) Several studies have beencarried out to characterize the importance of its ATP binding and hydrolysisactivities From these studies, it appears that the Walker A motif (nucleotidebinding) may be important for Cdc6 binding to chromatin, and Walker B mo-tif (nucleotide hydrolysis) is involved in MCM2-7 loading (Herbig et al., 1999;Perkins and Diffley, 1998; Weinreich et al., 1999) In addition to replicationdefects caused by mutation in the Walker A and B regions, certain mutations

contain-in the sensor regions are also detrimental to replication, often by failcontain-ing torecruit MCM (Schepers and Diffley, 2001) These studies indicate the ATPbinding and hydrolysis of Cdc6 are critical to its function in many differentorganisms

In addition to its direct regulation, which serves to limit replication toonce, and only once per cell cycle by controlling MCM loading, Cdc6 alsohas several other secondary roles Interestingly, Cdc6 seems to regulate ORC

by inhibiting its non-specific DNA binding (Harvey and Newport, 2003;Mizushima et al., 2000) By increasing sequence specificity of ORC, Cdc6 may

be playing an indirect role in origin selection, especially in higher otes where consensus initiators have been elusive This function may also helpprevent inefficient fork firing by ensuring ORCs are directed to specific sites,presumably spaced evenly along the chromosome

eukary-Cdc6 is not only regulated by the cell-cycle; it is also cleaved or degradedduring apoptosis (Blanchard et al., 2002; Pelizon et al., 2002) It is not entirelyclear why Cdc6 would be a target for apoptotic machinery; the cell no longerneeds to worry about proper replication, as it is dying However, this loss ofCdc6 may be part of the programmed cell death, halting replication initiation

in preparation for DNA fragmentation This intriguing finding illustrates theimportance of Cdc6 in replication, and thus, the advantage of being able totightly regulate its function in pre-RC formation

MCM2-7

The MCM (mini chromosome maintenance) genes were originally identified

in several independent screens as mutants having cell cycle defects, or chromosome perpetuation defects [reviewed in (Dutta and Bell, 1997)] These

mini-proteins were found to be the complex in Xenopus responsible for licensing,

a regulatory activity that allows cells to replicate in S-phase, and not againuntil an intervening mitosis occurs (Chong et al., 1995; Kubota et al., 1997;Madine et al., 1995; Thommes et al., 1997) As mentioned earlier, MCM2-7complex requires the chromatin loading of Cdt1 and Cdc6 (and thus, ORC)for its own chromatin loading Although MCM2-7 binding depends on ORC,Cdc6, and Cdt1, once the complex is loaded onto chromatin, ORC and Cdc6are no longer required for DNA replication This suggests ORC and Cdc6 act

to load MCM2-7, but not to maintain this chromatin association (Hua andNewport, 1998; Rowles et al., 1999)

The chromatin loading of MCM2-7 is regulated in a complex and dant manner Its additional role as the replication licensing factor illustratesits importance to replication as a whole As such, its function has long been

redun-an intriguing mystery MCM2-7 seemed to colocalize with DNA polymeraseε

using ChIP (Aparicio et al., 1997; Zou and Stillman, 2000) and is criticalfor replication elongation in vivo (Labib et al., 2000) Early bioinformaticsanalysis suggested the MCMs may be involved in strand opening (Koonin,1993) Biochemical analysis of the proteins confirmed this The mammalianMCM4,6,7 complex was purified, and this complex was found to have a mod-erate helicase (DNA unwinding) activity (Ishimi, 1997; You et al., 1999) Thefission yeast MCM4,6,7 complex also has helicase activity (Lee and Hurwitz,2000) Interestingly, when MCM2 or MCM3,5 were present in the complex,helicase activity was lost (Lee and Hurwitz, 2000; Sato et al., 2000; You et al.,1999) An archaeal MCM protein has been identified, and also has a helicaseactivity (Chong et al., 2000; Kelman et al., 1999; Shechter et al., 2000) Electronmicroscopy studies of the MCM complex indicate that they form a heterohex-amer ring structure (Adachi et al., 1997; Sato et al., 2000) This is supported by

a recent report which proposes double head-to-head hexamers from a tal structure of an archaeal MCM (Fletcher et al., 2003) This structure sharessimilar features with that of T-antigen, suggesting a common function to un-wind double strand DNA (Li et al., 2003a) Despite the inhibitory nature ofMCM2, 3, and 5, they, like MCM4,6,7, are essential in yeasts [for review see(Dutta and Bell, 1997; Kelly and Brown, 2000)] This puzzling result seems

crys-to indicate that MCM4,6,7 serves as the catalytic helicase domain, while theother subunits act to modulate MCM activity The exact mechanism of suchregulation is still unknown

Like certain ORC subunits and Cdc6, each member of the MCM2-7 plex has ATPase activity This ATPase activity is critical for viability; mutants

com-in the Walker A domacom-ins show S-phase defects and cell cycle arrest (Schwachaand Bell, 2001) Biochemical analysis of MCM helicase activity showed thatthe ATPase activity of these proteins is required for the helicase activity(Ishimi, 1997; Lee and Hurwitz, 2000; You et al., 1999) These studies indicatedthat the helicase activity of these proteins is most likely the in vivo func-tion required for replication Interestingly, Walker A mutations in MCM4,6,7are much more toxic to yeast than similar mutations in MCM2,3,5 AlthoughMCM2,3,5 are actually inhibitory for helicase activity (as described above),they are required for optimal ATPase activity of the complex (Schwacha andBell, 2001) This further supports the role of the MCM4,6,7 complex as thecatalytic domain, and MCM2,3,5 serving a regulatory function.

The chromatin association of MCM2-7 is regulated by several othermethods to control replication In budding yeast, MCM2-7 is exported fromthe nucleus beginning in S-phase (Labib et al., 1999; Nguyen et al., 2000)

In higher eukaryotes, this behavior has not been described However, pus egg extract MCMs dissociate from the chromatin as S-phase progresses

Xeno-(Kubota et al., 1997; Thommes et al., 1997) In humans, MCMs also seem todissociate during S-phase, and reassociate in late G2/M-early G1 (Mendez

and Stillman, 2000) This dissociation could be a critical step to prevent replication Once MCM complex is removed, it cannot reassociate until anintervening mitosis occurs (Seki and Diffley, 2000) This aspect of MCM reg-ulation led it to be thought of as the critical factor necessary for chromatinlicensing Once it is removed from chromatin, it requires the activity of ORC,Cdc6 and Cdt1 to be reloaded However, these proteins are unable to reloadMCM in S-phase; they are held inactive by Cdk activity through variousmechanisms described above Therefore, in order to reload MCM, a period oflow Cdk activity must be reached (in G1) to activate these proteins, allowingthem to reload the MCM2-7 complex

re-3.5

Geminin

Geminin is a small protein originally identified in a search for proteins

degraded by mitotic Xenopus egg extracts (McGarry and Kirschner, 1998).

Characterization of this protein indicated it was cell cycle regulated, withmaximum expression occurring in S/G2, followed by degradation in mitosis.

Contrary to other replication factors discussed here, geminin inhibits cation by preventing loading of MCM2-7 complex It was subsequently deter-mined that geminin binds Cdt1, thus inhibiting replication This inhibitionwas rescued by the addition of excess Cdt1 (Tada et al., 2001; Wohlschlegel

repli-et al., 2000) In vitro evidence demonstrates that geminin binding to Cdt1 maydisrupt an interaction between Cdt1 and MCM6 (Yanagi et al., 2002) The dis-ruption of MCM6 binding may be preventing the recruitment of the MCM2-7complex Additionally, the same study suggested that Cdt1 can bind DNA,

and this interaction was also disrupted by geminin inhibition A more cent study suggests that a Cdt1 binding activity to MCM2 and to Cdc6 is alsoinhibited by geminin binding (Cook et al., 2004) Structural studies indicatethat geminin forms a coiled-coil dimer which, together with an N-terminalflexible portion, executes a bipartite interaction with Cdt1 (Lee et al., 2004;Saxena et al., 2004) Initiation inhibition by geminin is now thought to be

re-a pre-art of the replicre-ation licensing system thre-at prevents multiple rounds of

replication in Xenopus egg extracts (Arias and Walter, 2004; Li and Blow,

2004) Although Cdt1 is the primary target of regulation, geminin provides

an additional pathway to inactivate Cdt1, and thus preventing ate replication In mammalian systems, overexpression of Cdt1 or depletion

inappropri-of geminin is sufficient to cause massive rereplication and checkpoint vation (Melixetian et al., 2004; Zhu et al., 2004) Abrogating this checkpointcauses mitotic distress and eventual cell death, demonstrating the importance

acti-of limiting replication to a single event per cell cycle

3.6

Summary

The pre-RC is composed of a variety of proteins that serve to recruit MCM2-7

to selected genomic regions, establishing a potential site for replication tiation (Fig 1) The chromatin loading of MCM2-7 depends on the loading

ini-of Cdt1 and Cdc6, which in turn depend on the loading ini-of ORC This

step-Fig 1 Regulation of the pre-RC The pre-RC functions to load MCM2-7 complex, the potential replicative helicase ORC binds chromatin initially at origins Its binding is re- quired for the chromatin association of Cdt1 and Cdc6, which are themselves required

to load the MCM2-7 complex This loading occurs in late mitosis until late G1 Upon entry into S-phase, increasing cyclin/Cdk activity inhibits the loading of MCM2-7 This

increased cyclin/Cdk activity promotes Orc1 and Cdc6 dissociation from chromatin,

and degradation of Cdt1 (and perhaps also Orc1 and Cdc6.) Additionally, expression of geminin, a protein inhibitory of Cdt1, increases in S-phase, further preventing MCM2-7 loading

wise assembly of factors is regulated at each stage, mostly through changes

in protein levels, chromatin affinities, or activity throughout the cell cycle.The activity of the pre-RC is limited to the time between late M and lateG1/early S This period is notable due to the absence of Cdk activity As many

pre-RC factors are inhibited by Cdk activity, they can only function to loadMCM2-7 in the late M to late G1 periods Once the cells pass into S-phase,cyclin/Cdk activity is high, and several pre-RC components dissociate from

the chromatin, or are degraded, and thus, are no longer able to load MCM2-7.Additionally, the inhibitor protein geminin is synthesized in early S-phase,further preventing any MCM loading by directly inhibiting Cdt1 These re-sults fit the original model of a “replication licensing” activity (Blow andLaskey, 1988) Early observation showed that once a given region of DNA hasreplicated, it cannot replicate again until an intervening mitosis occurs Itnow appears that MCM2-7 loading is the licensing activity, as it is requiredfor replication, and yet the loading cannot happen once replication has be-gun This is due to the inhibition of the pre-RC components by cyclin/Cdk

activity and geminin These regulatory mechanisms ensure that licensing(MCM2-7 loading) can only occur once in each cell cycle before replication.Intriguingly, viral episomes subjected to similar once-per-cell cycle replica-tion control, like Epstein-Barr-Virus derived episomes, also appear to use thecellular initiation factors for origin licensing (Chaudhuri et al., 2001; Dhar

et al., 2001b; Schepers et al., 2001)

Several recent studies have helped determine a likely function for Mcm10

In Xenopus egg extracts and fission yeast, Mcm10 is loaded onto chromatin

in an MCM2-7 dependent manner, and is itself required to load Cdc45 inG1/early S [discussed below] (Gregan et al., 2003; Wohlschlegel et al., 2002b).

In budding yeast, the role of Mcm10 is not as clear Earlier studies point to

a role for Mcm10 in loading the MCM2-7 complex (Homesley et al., 2000),whereas a more recent study finds the opposite: MCM2-7 is required to loadMcm10 (Ricke and Bielinsky, 2004) Additionally, Mcm10 has been reported

to load the pre-initiation kinase Dbf4/Cdc7, which is required to activate

MCM2-7 [see below] (Lee et al., 2003) Mcm10 may also have a role in loadingthe polymerase complex as well It was recently shown that Mcm10 stabilizesmembers of the Pol-α primase complex, and is important for chromatin load-

ing of these subunits (Ricke and Bielinsky, 2004) Work in human cells hasrevealed a regulatory mechanism to control Mcm10 function In G2, Mcm10

is phosphorylated, released from the chromatin, and subsequently degraded

by the proteasome It reappears and rebinds chromatin in late G1/early S

(Izumi et al., 2001) Together, these data point to a role for Mcm10 in tion through a variety of potential mechanisms While past work has revealedmuch about the importance of Mcm10 in replication, many of the details con-cerning these diverse functions remain to be worked out

replica-4.2

Cdc45

Cdc45 was identified in a yeast screen for cold sensitive cell cycle mutants(Moir et al., 1982) Cdc45 has been shown to interact genetically with mem-bers of the MCM2-7 complex, and mutants show replication defects (Hen-nessy et al., 1991; Hopwood and Dalton, 1996; Zou et al., 1997) Supporting

a role in replication, Cdc45 has been found to bind chromatin in G1 and lease in S-phase Interestingly, this binding requires cyclin/Cdk activity (Zou

re-and Stillman, 1998) Such a requirement implies a separation from earlierinitiation stages; those steps leading up to loading of the MCM2-7 complexare negatively regulated by cyclin/Cdk phosphorylation Pre-RCs can only be

loaded in the absence of Cdk activity, at which point Cdc45 cannot be loaded.Only when cyclin/Cdks become active can Cdc45 be loaded and replication

begin

In addition to cyclin/Cdk regulation, Cdc45 also cooperates with Dbf4/

Cdc7 kinase complex to bind chromatin (see below) These proteins seem

to require each other for activating replication (Owens et al., 1997; Zou andStillman, 2000) As mentioned earlier, Cdc45 loading requires Mcm10 load-ing (Gregan et al., 2003; Wohlschlegel et al., 2002b) The activating function

of Cdc45 may be the chromatin loading of polymeraseα, which was shown to depend on Cdc45 chromatin loading in Xenopus extracts (Mimura and Taki-

sawa, 1998) In addition to its requirement in Pol-α loading, Cdc45 and RPA

(single strand binding protein complex) depend on each other for chromatinbinding (Zou and Stillman, 2000) This co-loading of Cdc45 and RPA mayaid origin unwinding, allowing Pol-α access to template DNA (Walter and

Newport, 2000) This is supported by data that show a Cdc45 requirement

for helicase activity of MCM2-7 and in vivo origin unwinding (Masuda et al.,2003; Pacek and Walter, 2004).

Recent work has discovered a novel protein that interacts with Cdc45 Thisprotein, Sld3, has been shown in budding yeast to bind chromatin with Cdc45

in G1 and S-phase This binding occurs at origins, suggesting a replicationinitiation role for Sld3 Expression of a mutant Sld3 inhibits the interactionbetween Cdc45 and MCM2, and prevents loading of RPA (Kamimura et al.,2001) Studies in fission yeast suggest that Sld3 has a role in loading Cdc45onto chromatin (Nakajima and Masukata, 2002; Yamada et al., 2004), and thatSld3 is also regulated by Dbf4/Cdc7 There is also evidence in these studies

that Sld3 mediates the interaction between Cdc45 and MCM2-7, further ing pre-RC formation with pre-IC loading Cdc45 appears to have many roles

link-in promotlink-ing replication link-initiation It will therefore be link-interestlink-ing to evaluatethe extent of regulation of Cdc45 by the cell-cycle and the response to DNAdamage

4.3

Dbf4/Cdc7

Dbf4/Cdc7 forms a regulator/kinase pair very similar to cyclin/Cdks Dbf4

association is required for the kinase activity of Cdc7, and Dbf4 itself is cellcycle regulated [for review see (Bell and Dutta, 2002; Masai and Arai, 2002)].Cdc7/Dbf4 mutants show replication defects in yeast Although early stud-

ies indicated a role in S-phase entry, more recent work shows the complex to

be important all throughout S-phase (Bousset and Diffley, 1998; Donaldson

et al., 1998) This is most likely due to a function for Dbf4/Cdc7 in activating

both early and late firing origins locally Dbf4/Cdc7 is thought to have

sev-eral potential roles in origin firing Dbf4/Cdc7 phosphorylates the MCM2-7

complex, both in vitro and in vivo (Kihara et al., 2000; Lei et al., 1997; hiro et al., 1999; Owens et al., 1997; Sato et al., 1997; Weinreich and Stillman,1999) This phosphorylation is thought to activate the MCM2-7 complex, al-lowing replication to proceed (Lei et al., 1997) Consistent with this, mutations

Os-in MCM5 (Bob1) allows yeast to bypass the requirement of Dbf4/Cdc7 (Hardy

et al., 1997) Additionally, Dbf4/Cdc7 is required for Cdc45 chromatin

asso-ciation, and subsequent loading of the replication machinery (Owens et al.,1997; Zou and Stillman, 2000) Although a specific mechanistic change re-sulting from phosphorylation is not known, Dbf4/Cdc7 seems to act as a late

stage signaling step to promote loading of the replication machinery

In order to control such a signal, Dbf4/Cdc7 is regulated in several ways.

The predominant regulatory mechanism is the requirement of Dbf4 for Cdc7kinase activity This allows the cells tight control; Dbf4 is cell cycle regulated,and so the kinase is only active at the appropriate time in the cell cycle Dbf4

is degraded by the APC in mitosis, and is resynthesized at G1/S, allowing

kinase activity to rise at the G1/S transition, and fall at the end of the cell

cycle (Brown and Kelly, 1999; Chapman and Johnston, 1989; Ferreira et al.,2000; Kumagai et al., 1999; Oshiro et al., 1999; Weinreich and Stillman, 1999).Even when Dbf4/Cdc7 is activated, its phosphorylation of MCM2-7 still re-

quires Mcm10 (Lee et al., 2003) By controlling the chromatin loading of earlyfactors, as well as the presence of activating regulators, the cell has many sig-naling pathways available to prevent DNA replication when conditions are notideal

Such unfavorable conditions would undoubtedly include DNA damage.When the template condition is poor, accurate duplication is a difficultprospect However, recent studies have revealed a mechanism to prevent lateorigin firing at a local level Dbf4 homologues in various organisms arehyperphosphorylated in response to hydroxyurea (HU) treatment (Brownand Kelly, 1999; Takeda et al., 1999; Weinreich and Stillman, 1999) The re-sponsible kinases were the Chk2 yeast homologues Rad53/Cds1, which are

implicated in DNA damage signaling This checkpoint mediated lation inhibits the kinase activity of Dbf4/Cdc7 in vitro (Kihara et al., 2000) Similarly, single strand gaps in Xenopus egg extracts activate an ATR medi-

phosphory-ated checkpoint that also inhibits Dbf4/Cdc7 activity (Costanzo et al., 2003).

The absence of kinase activity is presumably responsible for the tion of Cdc45 from the chromatin This link between checkpoint pathwaysand replication initiation stops a potentially damaging replication cycle fromoccurring

dissocia-4.4

GINS

Recent work from two independent studies has identified a novel proteincomplex required for replication initiation In one study, a combination of se-quential multicopy suppressor screens (in an Sld5 mutant background) andco-immunoprecipitations identified a complex of four proteins; Sld5, Psf1(partner of sld five), Psf2 and Psf3 This complex was termed GINS, short forthe numbers 5,1,2,3 in Japanese [Go, Ichi, Nii, San] (Takayama et al., 2003)

A similar complex was also purified from Xenopus egg extracts (Kubota et al.,

2003) In a separate screen using an inducible degron system, novel tion deficient mutants were identified (Kanemaki et al., 2003) The identifiedproteins turned out to be the same four proteins constituting GINS

replica-All three studies show that GINS are critical for replication The complexassociates with chromatin, and travels with the replication fork (Kanemaki

et al., 2003) Additionally, GINS chromatin association requires S-Cdk ity, similar to other pre-initiation complex members (Kanemaki et al., 2003;Kubota et al., 2003) However, licensing plays a role to restrict GINS activity,

activ-as it requires pre-RC loading for its own chromatin binding This chromatinbinding is also co-dependent with Cdc45, Dpb11, and Sld3; binding of any of