Regulation of S Phase

Results Probl Cell Differ (42) 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. Teer 1,2 · Anindya Dutta 1,2 (✉) 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. 1 Introduction DNA replication is fundamentally critical, and yet rather problematic for all life. Cells must be prepared to replicate the entire genome, and must do so in a concerted, rapid, and efficient fashion. Failures in this process not only yield potentially damaging mutations, but may also hinder proper genome segregation between offspring. This problem becomes even more pronounced with increasing size of a given genome. Cells have a variety of mechanisms to ensure that the proper environment exists to carry out replication. These mechanisms can be divided into proper identification of appropriate origins of replication, and subsequent loading of the replication machinery itself. We will focus our discussion on the initiation of replication in eukaryotes, and how it can be used by the cells to control S-phase progression. 32 J.K. Teer · A. Dutta 2 Origins of Replication 2.1 Genome Replicator Sequences Theoretically, replication would be carried out most efficiently by starting from 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,andwas 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 bp consensus seqeunce (A element) and several other B elements (Marahrens and Stillman, 1992). These replicator sequences were used later to identify the putative 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 were found in other ars regions (Dubey et al., 1996). Although the yeasts seem to have high sequence conservation from one replicator to the next, determining such 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 se- quence specificity in replicating regions (Hyrien and Mechali, 1992; Hyrien and Mechali, 1993; Mahbubani et al., 1992). Recent studies show that while the ORC proteins may prefer AT rich DNA stretches, they show no preference between defined origin sequences and control sequences in vitro, even with varying 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 et al., 1995). In early stages, origin selection was random, but when rDNA gene expression began in late blastula and early gastrula stages, initiation frequency 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 in the G1 phase also failed to initiate specifically (Wu and Gilbert, 1996). Taken Regulation of S Phase 33 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 in chinese 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 DHFR gene 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 of sequence specificity (Burhans et al., 1990; Kobayashi et al., 1998). Recently, a study using early labeled fragment hybridization (ELFH) showed that the earliest nascent strands could hybridize to many clones from different areas along the intergenic region, suggesting that replication can be initiated from many different sites (Dijkwel et al., 2002). Additionally, a deletion mapping experiment showed that replication could initiate from the intergenic region in the absence of the major sites, and even in the absence of 90%oftheregion (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 by other factors. Like Chinese hamster cells, few origins of replication are known in human cells. One of the earlier defined origins of replication is at the β-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 be present in this region to direct origin firing. Furthermore, an ectopically in- serted β-globin locus promoted initiation at the ectopic site (Aladjem et al., 1998). Deletion mapping of the β-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 lo- cus [5.8 kb] (Altman and Fanning, 2004), and the c-myc locus [2.4 kb] (Liu et al., 2003): ectopically inserted sequences can confer origin activity, and deletion of specific elements eliminates such activity. Unfortunately, the se- quence elements do not seem to be identical, and no consensus sequences have 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, an essential 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 origin firing by PCR of nascent strands, while studies supporting sequence inde- 34 J.K. Teer · A. Dutta pendent origin firing use ELFH and two dimensional gel electrophoresis. The possibility exists that different methodologies may have different effects on the results. In addition to sequence effects, many studies have implicated transcrip- tion in selection of replication origins. In the DHFR locus, transcription of DHFR itself is required for origin firing activity, and yet origins do not fire in the gene (Kalejta et al., 1998; Saha et al., 2004). In yeast, evidence exists for transcriptional correlation with replication (Muller et al., 2000), but this may 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 link between early origin firing and transcriptional activity by looking at replica- tion of developmentally regulated genes, as well as genes from asymmetrically active alleles [reviewed in (Goren and Cedar, 2003)]. In the latter case, the active 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 induced hyperacetylation 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 the open chromatin environment for transcription would also benefit replication, linking the two different activities. Although controversy exists as to whether higher eukaryotic origins are sequence 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 primary determinant of origin locations. However, as organism complexity increases, so does the complexity of origin determination. In metazoans, a consensus origin sequence has not yet been identified. Many reports show that certain sequence elements are important for firing, but that these elements for the most 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 elem- ents exist to provide a favorable environment for initiator loading. Other factors may also affect chromatin structure, especially during transcription. The positive correlation between origin firing and transcriptional activity in higher eukaryotes suggests that the more open chromatin structure not only allows efficient transcription, but efficient replication initiation as well. It is also plausible that different origins may have differential influences on their activity. Nearby transcriptional activity may be important for one origin, whereas critical sequence elements are important for another. In summary, Regulation of S Phase 35 origin selection in eukaryotes is defined by the proper environment for ini- tiator binding, whether defined solely by DNA sequence elements, structural elements, chromatin structure itself, or a combination of effects. Thus, the 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 subunit complex termed ORC, or Origin Recognition Complex (Bell and Stillman, 1992). Mutations in the A element that prevent ORC binding also prevent replication 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 is dependent 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. cere- visiae and S. pombe. Work in the latter organism has revealed that Orc4 dictates the specificity via a newly defined AT hook region (Kong and De- Pamphilis, 2001; Lee et al., 2001). ORC from higher eukaryotes, however, does not seem to have the same sequence specificity. Drosophila ORC showed lit- tle preference for chorion gene sequences compared to controls, but showed a much better preference for negative supercoiled DNA compared to relaxed or linear, suggesting secondary structure is more important than sequence for 36 J.K. Teer · A. Dutta initiator/replicator interactions in metazoans (Remus et al., 2004). Similarly, human ORC shows no preference for known origins compared to random DNA sequences, but does show a slight preference for AT rich DNA (Vashee et al., 2003). While ORC may be responsible for DNA binding, the mechan- 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 Xenopus egg extracts have demonstrated that ORC is required to load Cdt1 and Cdc6, themselves factors required for replication initiation [discussed further below] (Coleman et al., 1996; Maiorano et al., 2000). This dependence of replication factor recruitment on ORC helps to explain the lethality of all ORC subunit deletions in yeast. As the foundation of replication initiation complexes, 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 once per cell cycle; when Orc2 and Orc6 phosphorylation site mutants were intro- duced with constitutively active Cdc6 and MCM proteins (see Sects. 3.3 and 3.4, 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 ensur- 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 the cells a reversible way to prevent replication firing. 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). Simi- larly, mammalian Orc1 is phosphorylated. In Chinese hamster ovary cells, Orc1 interacts with cyclin A/Cdk1, which leads to the phosphorylation of Orc1. Inhibiting this phosphorylation with drugs allows Orc1 to rebind chro- matin, indicating that phosphorylation is important for chromatin release in mitosis (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). 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. In hamster cells, Orc1 seems to be mono- and di-ubiquitinated, which causes its release from chromatin in S-phase until M-G1 (Li and DePamphilis, 2002). Regulation of S Phase 37 In humans, several studies show that Orc1 is polyubiquitinated and then de- 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- 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 simply result from differences between organisms or even cell types, Orc1 binding to chromatin can be regulated in higher eukaryotes. This regulation allows the cells to control replication initiation at the basic level of the ORC, ensuring inappropriate replication initiation has little, if any, chance of success. 3.2 Cdt1 Cdt1 was first identified as a Cdc10 regulated gene in S. pombe.Thisgenewas found to be cell cycle regulated, and important for replication (Hofmann and Beach, 1994). It associates with Cdc6 and is required for loading of the MCM complex in several model systems (Maiorano et al., 2000; Nishitani et al., 2000; Tanaka and Diffley, 2002). Its own chromatin loading is dependent on ORC (Maiorano et al., 2000), supporting the idea that ORC serves as a foun- dation that recruits downstream factors for replication. Cdt1 protein itself is regulated 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 mas- sive re-replication is observed (Zhong et al., 2003). In humans, the SCF Skp2 complex is implicated in the destruction of Cdt1, and is dependent upon phosphorylation by cyclin-dependent kinases [Cdks] (Li et al., 2003b; Sugi- moto et al., 2004), although mutations in Cdt1 that disrupt association with Skp2 still permit degradation of Cdt1 in S-phase (Takeda et al., 2005). Re- cent work in Xenopus egg extracts indicates that the degradation of Cdt1 after replication initiation, together with its inhibition by the protein gemi- nin (discussed later), limit replication to a single round per cell cycle (Arias and Walter, 2004; Li and Blow, 2004). As Cdt1 is required for pre-RC loading, its inhibition by either geminin interaction or degradation will help prevent further pre-RC formation, and thus, further replication initiation. Consistent with this, overexpression of Cdt1 alone leads to extensive re-replication in human cells (Vaziri et al., 2003). In addition to its function as a replication licensing factor, Cdt1 has re- cently been implicated in preventing replication initiation after DNA damage in human cells. Cdt1 levels were found to be profoundly decreased after UV irradiation, and an E3 ligase, Cul4A-Roc1-Ddb1, was responsible for signaling 38 J.K. Teer · A. Dutta this degradation via the proteasome (Higa et al., 2003; Hu et al., 2004). In- terestingly, a separate study implicated the SCF Skp2 complex in the radiation induced degradation of Cdt1 (Kondo et al., 2004). It remains to be resolved which ubiquitin ligase is primarily responsible for both the cell cycle depen- dent modifications and the DNA damage induced modifications. 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 an early 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 for MCM 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 also found 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 overex- pression in S. pombe resulted in severe rereplication. Additionally, its protein levels cycle, with maximum levels present during the G1/S transition (Muzi- Falconi et al., 1996; Nishitani and Nurse, 1995). Cdc18 is phosphorylated upon entry into S-phase, causing its rapid degradation (Jallepalli et al., 1997). Not only 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 for MCM2-7 loading, thus implicating a sequential assembly of pre-RC compo- nents (Coleman et al., 1996). Similar results were observed in a human cell free 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 for promoting 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 Regulation of S Phase 39 after replication has initiated (Petersen et al., 1999). Some evidence exists that human Cdc6 is degraded in Sphase, just as in yeast, a point that will need resolution (Coverley et al., 2000; Mendez and Stillman, 2000). Recently it was also reported that although exogenous Cdc6 is exported from the nucleus in S-phase, endogenous Cdc6 is not (Alexandrow and Hamlin, 2004). One fur- ther study demonstrates that although Cdc6 is released from chromatin and then degraded, it is constantly being resynthesized and immediately binds chromatin, replacing molecules which were displaced (Biermann et al., 2002). This observation may reconcile earlier observed differences, allowing Cdc6 to bind 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, contain- ing Walker A and B motifs, and well as several sensor regions thought to detect nucleotide binding status (Liu et al., 2000). Several studies have been carried out to characterize the importance of its ATP binding and hydrolysis activities. From these studies, it appears that the Walker A motif (nucleotide binding) 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 replication defects caused by mutation in the Walker A and B regions, certain mutations in the sensor regions are also detrimental to replication, often by failing to recruit MCM (Schepers and Diffley, 2001). These studies indicate the ATP binding and hydrolysis of Cdc6 are critical to its function in many different organisms. In addition to its direct regulation, which serves to limit replication to once, and only once per cell cycle by controlling MCM loading, Cdc6 also has 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 eukary- otes where consensus initiators have been elusive. This function may also help prevent inefficient fork firing by ensuring ORCs are directed to specific sites, presumably spaced evenly along the chromosome. Cdc6 is not only regulated by the cell-cycle; it is also cleaved or degraded during apoptosis (Blanchard et al., 2002; Pelizon et al., 2002). It is not entirely clear why Cdc6 would be a target for apoptotic machinery; the cell no longer needs to worry about proper replication, as it is dying. However, this loss of Cdc6 may be part of the programmed cell death, halting replication initiation in preparation for DNA fragmentation. This intriguing finding illustrates the importance of Cdc6 in replication, and thus, the advantage of being able to tightly regulate its function in pre-RC formation. 40 J.K. Teer · A. Dutta 3.4 MCM2-7 The MCM (mini chromosome maintenance) genes were originally identified in several independent screens as mutants having cell cycle defects, or mini- chromosome perpetuation defects [reviewed in (Dutta and Bell, 1997)]. These 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 again until an intervening mitosis occurs (Chong et al., 1995; Kubota et al., 1997; Madine et al., 1995; Thommes et al., 1997). As mentioned earlier, MCM2-7 complex 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 Cdc6 are no longer required for DNA replication. This suggests ORC and Cdc6 act to load MCM2-7, but not to maintain this chromatin association (Hua and Newport, 1998; Rowles et al., 1999). The chromatin loading of MCM2-7 is regulated in a complex and redun- dant manner. Its additional role as the replication licensing factor illustrates its importance to replication as a whole. As such, its function has long been an intriguing mystery. MCM2-7 seemed to colocalize with DNA polymerase ε using ChIP (Aparicio et al., 1997; Zou and Stillman, 2000) and is critical for replication elongation in vivo (Labib et al., 2000). Early bioinformatics analysis suggested the MCMs may be involved in strand opening (Koonin, 1993). Biochemical analysis of the proteins confirmed this. The mammalian MCM4,6,7 complex was purified, and this complex was found to have a mod- erate helicase (DNA unwinding) activity (Ishimi, 1997; You et al., 1999). The fission 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 helicase activity (Chong et al., 2000; Kelman et al., 1999; Shechter et al., 2000). Electron microscopy 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 crys- tal structure of an archaeal MCM (Fletcher et al., 2003). This structure shares similar features with that of T-antigen, suggesting a common function to un- wind double strand DNA (Li et al., 2003a) Despite the inhibitory nature of MCM2, 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 to indicate that MCM4,6,7 serves as the catalytic helicase domain, while the other subunits act to modulate MCM activity. The exact mechanism of such regulation is still unknown. Like certain ORC subunits and Cdc6, each member of the MCM2-7 com- plex has ATPase activity. This ATPase activity is critical for viability; mutants [...]... of cases, these proteins were either both overexpressed or both normal Interestingly, in cases showing Cdc6 and Cdt1 overexpression, p53 status affected the ploidy of the cells When p53 was mutated, 73% of the cases showed aneuploidy, compared to only 28% of cases with wild-type p53 This result demonstrates that p53 can protect against the damaging effects of high levels of Cdt1 and Cdc6 and agrees.. .Regulation of S Phase 41 in the Walker A domains show S- phase defects and cell cycle arrest (Schwacha and Bell, 2001) Biochemical analysis of MCM helicase activity showed that the ATPase activity of these proteins is required for the helicase activity (Ishimi, 1997; Lee and Hurwitz, 2000; You et al., 1999) These studies indicated that the helicase activity of these proteins is most likely... Regulation of S Phase 49 distinct sites in the genome Once this is accomplished, the replication machinery is loaded, and replication begins Each step in this process is subject to regulation, allowing the cell to halt replication under a wide variety of adverse conditions, including DNA damage 5 S- phase Regulation and Cancer Proper reproduction of the genome is critical for viability Mutations and mistakes... humans, MCMs also seem to dissociate during S- phase, and reassociate in late G2/M-early G1 (Mendez and Stillman, 2000) This dissociation could be a critical step to prevent rereplication Once MCM complex is removed, it cannot reassociate until an intervening mitosis occurs (Seki and Diffley, 2000) This aspect of MCM regulation led it to be thought of as the critical factor necessary for chromatin licensing... has recently made great progress, there are many questions yet to be answered Although the general licensing system has been described, more detailed mechanistic problems still abound How does ORC recognize origins? How are origins defined in humans? How do the members of pre-RC interact to load MCM2-7? MCM27 itself is still somewhat mysterious, as its in vitro helicase activity is only apparent as... initiation In one study, a combination of sequential multicopy suppressor screens (in an Sld5 mutant background) and co-immunoprecipitations identified a complex of four proteins; Sld5, Psf1 (partner of sld five), Psf2 and Psf3 This complex was termed GINS, short for the 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... levels, targeting these proteins may decrease viral load at concentrations that have minimal effects on the chromosomal replication of cells Many other viruses are implicated in cancer, and some of these may also utilize host replication initiation factors For example, Kaposi s Sarcoma-Associated herpesvirus (KSHV) was shown to utilize ORC for latent replication (Stedman et al., 2004) Therefore, studies... inhibitory of Cdt1, increases in S- phase, further preventing MCM2-7 loading Regulation of S Phase 43 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 late G1/early S This period is notable due to the absence of Cdk activity As many... chromatin association of MCM2-7 is regulated by several other methods to control replication In budding yeast, MCM2-7 is exported from the nucleus beginning in S- phase (Labib et al., 1999; Nguyen et al., 2000) In higher eukaryotes, this behavior has not been described However, Xenopus egg extract MCMs dissociate from the chromatin as S- phase progresses (Kubota et al., 1997; Thommes et al., 1997) In humans,... Mills AD, Stoeber K, Marr J, Laskey RA, Coleman N (1998) Improved cervical smear assessment using antibodies against proteins that regulate DNA replication Proc Natl Acad Sci USA 95:14932–14937 Williams RS, Shohet RV, Stillman B (1997) A human protein related to yeast Cdc6p Proc Natl Acad Sci USA 94:142–147 Wilmes GM, Archambault V, Austin RJ, Jacobson MD, Bell SP, Cross FR (2004) Interaction of the S- phase . chromatin as S- phase progresses (Kubota et al., 1997; Thommes et al., 1997). In humans, MCMs also seem to dissociate during S- phase, and reassociate in. mutants Regulation of S Phase 41 in the Walker A domains show S- phase defects and cell cycle arrest (Schwacha and Bell, 2001). Biochemical analysis of MCM